UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Instability of G-rich DNA in Caenorhabditis elegans Cheung, Iris 2005

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
831-ubc_2005-104767.pdf [ 18.66MB ]
Metadata
JSON: 831-1.0092324.json
JSON-LD: 831-1.0092324-ld.json
RDF/XML (Pretty): 831-1.0092324-rdf.xml
RDF/JSON: 831-1.0092324-rdf.json
Turtle: 831-1.0092324-turtle.txt
N-Triples: 831-1.0092324-rdf-ntriples.txt
Original Record: 831-1.0092324-source.json
Full Text
831-1.0092324-fulltext.txt
Citation
831-1.0092324.ris

Full Text

INSTABILITY OF G-RICH D N A IN CAENORHABDITIS E L E G A N S  by IRIS C H E U N G  B . S c , The University of British Columbia, 2000  A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in  THE F A C U L T Y OF G R A D U A T E STUDIES (Medical Genetics)  T H E UNIVERSITY OF BRITISH C O L U M B I A June 2005  © Iris Cheung, 2005  ABSTRACT  Mutations in genes that function in maintaining genome stability, such as those involved in D N A replication, D N A repair, or cell-cycle checkpoints, may lead to extensive genetic alterations, leading to the so-called "mutator phenotype". In Caenorhabditis elegans, the mutant strain dog-l(gklO) displays such a phenotype. Molecular characterization of the strain revealed genome-wide deletions involving a very specific type of repeat, consisting of polyG tracts paired with poly-C tracts ((G/C)n). Deletions have unique structural characteristics and only occurred in roughly half of the (G/C)n tracts examined. DOG-1 contains the seven signature motifs of a DExH-box helicase. Based on these observations, a model was proposed in which the putative helicase DOG-1 is required for unwinding secondary structures formed by G-rich D N A during lagging strand synthesis. In the absence of functional DOG-1 such secondary structures may lead to deletions via an unknown mechanism. Because telomeric D N A is capable of forming secondary structure in vitro, dogl(gklO) was examined for telomere defects. To measure telomere length in C. elegans with higher sensitivity and accuracy, a PCR-based technique, called S T E L A was adapted to C. elegans thereby enabling chromosome-specific telomere length measurement from as few as a single worm. Telomere length analysis using this technique revealed the presence of short telomeres that were clearly distinct from the bulk telomere length distributions in different wild-type strains. This suggests that processes other than end-replication losses and telomerase-mediated lengthening contribute to telomere length heterogeneity in C. elegans. A n increased frequency of such short outlying telomeres was observed in the telomerase mutant trt-1, indicating that besides replicative loss, telomerase is also required for preventing large scale loss of telomeric D N A . Analysis of telomere length in dog-l(gklO) using S T E L A showed no significant shortening of average telomere length or increased frequency of short telomeres. Therefore, DOG-1 appeared to be required specifically for the maintenance of (G/C)n tracts within the genome.  ii  T A B L E OF CONTENTS  Abstract  ii  Table of contents  iii  List of tables  vii  List of figures  viii  List of abbreviations  x  Acknowledgements  xiv  C H A P T E R ONE: Introduction 1.1 Maintenance of repeat sequences  2  1.1.1 Distribution of microsatellites in eukaryotic genomes  2  1.1.2 Microsatellite instability in  3  1.1.3 Mechanisms of changes in tract length in microsatellites  5  1.1.4 Parameters affecting stability of microsatellites  6  1.1.5 Secondary D N A structures formed by microsatellites  7  1.1.6 G-quadruplex  10  1.1.6.1 Potential involvement of G-quadruplexes in biological processes  10  1.1.6.2 Implications of the G-quadruplex structure on gene expression  11  1.1.6.3 Implications of the G-quadruplex structure on recombination  12  1.1.6.4 Evidence of G-quadruplex formation at telomeres  13  1.2 Helicases and genome maintenance  14  1.2.1 Structure and classification of helicases  14  1.2.2 X P B a n d X P D  17  1.2.3 RecQ  17  1.2.4 BACH1/BRIP1  19  1.3 Repeat sequences at chromosome ends  20  1.3.1 TelomericDNA  22  1.3.2 Structure at telomere termini  24  1.3.3 Higher order structure of telomeres  26  1.3.4 Telomere protection  28  iii  1.3.5 The telomerase ribonucleoprotein complex  33  1.3.6 Consequences of telomerase deficiency  37  1.3.7 Telomere length regulation  42  1.4 G-rich D N A in C. elegans  44  1.4.1 C. elegans as a model organism  44  1.4.2 Features of (G/C)n in the C. elegans genome  45  1.4.3 Microsatellite instability in C. elegans mutants  47  1.4.4 Telomere defects in C. elegans mutants  47  1.5 Thesis objectives  49  C H A P T E R TWO: dog-1 is required for the maintenance of G-rich D N A in C. elegans 2.1 Introduction  50  2.2 Materials and Methods  50  2.2.1 Strains  50  2.2.2 Worm lysis  51  2.2.3 Genotyping of dog-1  51  2.2.4 Measurement of forward mutation rate in dog-1 (gklO) by the eTl translocation  51  2.2.5 Detection of deletions  53  2.2.6 R N A i silencing of dog-1 expression  53  2.3 Results  54  2.3.1 gklO is a deletion allele of dog-1 and confers a mutator phenotype  54  2.3.2 Frequent deletion of a (G/C) o within vab-1 in dog-l(gklO)  58  2  2.3.3 Frequent deletion of a  (G/C) 6 2  within the cosmid F55F3 in dog-l(gklO).... 60  2.3.4 Somatic tissues are susceptible to deletions in dog-1 (gklO)  61  2.3.5 R N A i silencing of dog-1 expression  63  2.3.6 Genome-wide deletions in dog-l(gklO)  65  2.3.7 A proposed model for deletions of (G/C)n in dog-1 (gklO)  66  2.4 Discussion  68  2.4.1 DOG-1 and its homologue in human  iv  68  2.4.2 Deletions in dog-l(gklO) rarely resulted in heritable mutations  69  2.4.3 Deletions of different (G/C)n occurred at different frequency  70  2.4.4 The model  71  2.4.5 Do (G/C)n have any function in the C. elegans!  73  C H A P T E R T H R E E : Single telomere length analysis (STELA) in C. elegans 3.1 Introduction  75  3.2 Materials and Methods  76  3.2.1 Bulk genomic D N A isolation and ligation  76  3.2.2 D N A isolation and ligation from 5 worms or single worms  77  3.2.3 Telomere amplification and detection  77  3.2.4 Oligonucleotides  78  3.3 Results and Discussion  78  3.3.1 Design of S T E L A  78  3.3.2 S T E L A from bulk genomic D N A  80  3.3.3 S T E L A from 5 worms or single worms  81  3.3.4 Use of S T E L A to measure telomere shortening in mutants  85  C H A P T E R FOUR: Use of S T E L A in studying telomere biology in C. elegans 4.1 Introduction  88  4.2 Materials and Methods  89  4.2.1 Strains  89  4.2.2 Genotyping of trt-1  89  4.2.3 S T E L A  89  4.2.4 Analysis of S T E L A data  90  4.3 Results  90  4.3.1 Fluctuations of telomere length in N2  90  4.3.2 Telomere length heterogeneity within clonal population  91  4.3.3 Evidence of tissue-specific telomere length  92  v  4.3.4 Variations in telomere length in wildtype C. elegans strains  94  4.3.5 Progressive telomere shortening in trt-l(ok410)  98  4.3.6 Reduced telomere length heterogeneity in trt-l(ok410)  100  4.3.7 No apparent telomere phenotype in dog-l(gklO)  103  4.4 Discussion  106  4.4.1 Telomere length variations in C. elegans  106  4.4.2 Presence of short outlying telomeres in wildtype strains  106  4.4.3 Reduced telomere length heterogeneity in telomerase mutant  107  4.4.4 Increased frequency of short outlying telomeres in trt-l(ok410)  109  4.4.5 Rate of telomere shortening in trt-l(ok410)  110  4.4.6 Absence of telomere defects in dog-l(gklO)  Ill  C H A P T E R FIVE: General discussion 5.1 Relation of DOG-1 to BACH1  112  5.2 Absence of significant telomere defects in dog-l(gklO)  113  5.3 Recommendations and considerations in using S T E L A  114  BIBLIOGRAPHY  118  Appendix A : Primers and PCR conditions used in amplification of (G/C)n  149  Appendix B : Genomic, spliced and protein sequences of gklO  150  Appendix C: Repeat sequences that are stable in dog-l(gklO)  153  Appendix D: Alternation in recombination frequencies in F25H2.13 (RNAi)  154  Appendix E : Telomere length measurement in dog-l(gkl0) and a rescued strain by terminal restriction fragment (TRF) analysis  156  vi  LIST OF T A B L E S  Table 1-1. Telomeric repeats in different organisms  23  Table 2-1. Frequency and size dependence of deletions involving (G/C)n tracts in dog-l(gklO)  67  Table 3-1. Distribution of telomere length in N2 and mrt-2  ,  87  Table 4-1. Distribution of telomere length in different wildtype C. elegans strains  97  Table 4-2. Progeny size in 16 lines oitrt-l(ok410)  101  Table 4-3. Rate of V L telomere shortening (kb/generation) in trt-l(ok410)  102  vii  LIST OF FIGURES  Figure 1-1. Model proposed by Kang et al (1995) to explain orientation-dependent expansions and deletions of (CTG)n in E. coli  8  Figure 1-2. The G-quadruplex structure  9  Figure 1-3. Classification of helicases  15  Figure 1-4. D N A replication is predicted to generate asymmetric chromosome ends  21  Figure 1-5. Higher order structure of telomeres  27  Figure 1-6. Proposed functions of Cdcl3p in telomere protection and telomere elongation. 32 Figure 1-7. Structure of the telomerase R N A is conserved  34  Figure 1-8. Modular structure of telomerase reverse transcriptase (TERT)  36  Figure 1-9. Telomere length is regulated by multiple proteins in humans  44  Figure 2-1. Intron-exon organization of dog-1 (F33H2.1) and \Xs gklO-mx\\ allele  54  Figure 2-2. Alignment of C. elegans DOG-1 and human BACH1/BRIP1  55  Figure 2-3. Alignment of C. elegans DOG-1 and F25H2.13 gene product  56  Figure 2-4. Variable brood size and mutator phenotype in dog-1 (gklO)  57  Figure 2-5. Sturecure of germline and somatic deletions involving vab-1 exon5 in dog-1(gklO)  59  Figure2- 6. Vab-1 exon5 was involved in deletion at a high frequency in dog-1 (gklO)  60  Figure 2-7. Disruption of dog-1 leads to frequent deletions involving the  (G/Q26  in  F55F3  61  Figure 2-8. Structure of deletions involving the  (G/Q26  in an intergenic region within the  cosmid F55F3  62  Figure 2-9. Size distribution of deletions involving F55F3 in dog-l(gkl0)  63  Figure 2-10. Haploinsufficiency in dog-1 heterozygotes  64  Figure 2-11. Somatic tissues are susceptible to deletions in dog-1 (gklO)  65  Figure 2-12. Inhibition of dog-1 expression using R N A i resulted in deletions similar to dog-1 (gklO)  66  viii  Figure 2-13. The invariable occurrence of breakpoints at the 5' end of the (C)n (i.e. the 3'end of the (G)n) is compatible with deletions resulting from the failure of lagging-strand D N A synthesis  72  Figure 3-1. Design of S T E L A in C. elegans  79  Figure 3-2. The fragment amplified using the 509 and 510 primers (509/510) contains a duplication that is not present in the previously published sequence of cTel3X  81  Figure 3-3. S T E L A from bulk N2 genomic D N A  82  Figure 3-4. Telomere length measurement from 5 worms by S T E L A  83  Figure 3-5. A single worm is sufficient for telomere length measurement by S T E L A  84  Figure 3-6. Telomere shortening in mrt-2  87  Figure 4-1. Telomere length fluctuations in wildtype C. elegans  91  Figure 4-2. Telomere length heterogeneity in wildtype C. elegans  92  Figure 4-3. Telomere length pattern in the presence or absence of germline  93  Figure 4-4. Variations in the length of V L telomeres in different wildtype C. elegans Strains  96  Figure 4-5. Progressive telomere shortening in trt-l(ok410)  100  Figure 4-6. Frequency of different amounts of telomeric D N A lost between generations  103  Figure 4-7. Reduced telomere length heterogeneity in trt-l(ok410)  104  Figure 4-8. No apparent telomere defects in dog-l(gklO)  105  Figure 5-1. Principle of P E T R A in telomere length measurement in Arabidposis  117  ix  LIST OF A B B R E V I A T I O N S  4NQO  4-nitroquinoline 1-oxide  aa  amino acid  BACH  B R A C 1 -associated C-terminal helicase  BAX  BCL2-associated X  BER  base excision repair  BLAST  basic local alignment search tool  bp  basepairs  BRCA  breast cancer  BrdU  bromodeoxyuridine  BRIP  B R C A 1 -interacting protein  BS  Bloom syndrome  CDC  cell division cycle  CFP  circular form of linear plasmid  CLK  clock  CO-FISH  chromosome orientation fluorescence in situ hybridization  CS  Cockane syndrome  DAP I  diamidinophenolindole  DAT  dissociates activities of telomerase  DBD  D N A binding domain  DMS  dimethyl sulfate  DNA  deoxyribonucleic acid  DOG  deletion of G  DPY  dumpy  DSB  double-stranded break  x  ES  embryonic stem  EST  ever shorter telomere  FEN  flap endonuclease  FMR  fragile X mental retarded  GI  gastrointestinal  GLP  abnormal germ line proliferation  GQN  G-quartet nuclease  HNPCC  heritable non-polyposis colorectal cancer  hnRNP  heterogeneous nuclear riboncleoprotein  HR  homologous recombination  IGFIIR  insulin-like growth factor II receptor  ILPR  insulin gene-linked polymorphic region  INV  invading strand  kb  kilobases  Mb  megabases  MEF  mouse embryonic fibroblast  MLH  MutL homolog  MMR  mismatch repair  MRT  mortal germline  MSH  MutS homolog  NER  nucleotide excision repair  NHE  nuclease hypersensitive element  NHEJ  non-homologous end-joining  NHL  novel helicase-like  NMR  nuclear magnetic resonance  nt  nucleotides  ORF  open reading frame  Po  Parental generation  PARP  poly(ADP-ribose) polymerase  PCNA  proliferating cell nuclear antigen  PCR  polymerase chain reaction  PD  population doublings  PETRA '  primer extension telomere repeat amplification  PIP  POT 1-interacting protein  PMS  post-meiotic segregation  POT  protection of telomere  PTOP  POT1- and TIN2-organizing protein  Q-FISH  quantitative fluorescence in situ hybridization  RAP  repressor/activator protein  RD  recruitment domain  RIF  Raplp-interacting factor  RNAi  R N A interference  RNP  ribonucleoprotein  RPA  replication protein A  RT  reverse transcriptase  RTEL  regulatore of telomere length  RTEL  regulator of telomere length  RTS  Rothmund-Thomson syndrome  SCE  sister chromatid exchange  SF  superfamily  xii  SGS  slow growth suppressor  siRNA  small interfering R N A  SSB  single-stranded binding protein  STELA  single telomere length analysis  STN  suppressor of cdc thirteen  TEN  telomeric pathway in association with stnl  TERT  telomerase reverse transcriptase  TF  transcription factor  TIN  TRF 1-interacting nuclear factor  TINT  TIN2-interactiing protein  TLC  telomerase component  TR  telomerase R N A  TRF  T T A G G G repeat binding factor  TRF  terminal restriction fragment  TRT  telomerase reverse transcriptase  TTD  tichothiodystrophy  UNC  uncoordinated  ws  Werner syndrome  XP  xeroderma pigmentosum  xiii  ACKNOWLEDGEMENTS  It is time to say thanks to the people who have contributed to my experience during the past 5 years:  Of course if my supervisors Peter Lansdorp and Ann Rose did not give me the opportunity, none of i f would have happened. I also wish to thank them for their guidance, support, encouragement, enthusiasm and ideas. Mike Schertzer has helped a lot technically, and he has many interesting insights and ideas which I wish to thank for. I also appreciate his willingness to share his bench with me in the old Research Center. Dale and Liz are great people to talk to in the lab. Liz has become known to me as the person who knows everything around the lab. Dale has taught me a lot about life in general from his personal experience. Many thanks to other members from the Lansdorp lab, past and present, for making the lab a decent place to work in, especially Irma, for organizing around the lab. I also wish to thank members in the Rose lab, past and present, especially Berdjis, who has been so helpful in making plates and keeping track of strains etc, and Vijhee, for the mutant strain that I continued to work on and led to my project.  I would like to thank my committee members Rob Kay and Steven Jones for their useful suggestions and involvement in my graduate training, David Baillie for his unpublished findings and interesting stories, Duncan Baird for his help with STELA, and Sara Selig for her valuable tips.  I would like to mention some good friends that I have made while working here, Pat, Gigi, and Jennifer, for all the fun outside of work. I could not possibly express how grateful I am having met Ping, who has opened my eyes to a completely different world. Most importantly, I wish to thank my parents, for being the best parents. I feel I owe everything to them.  xiv  To my parents,  xv  C H A P T E R ONE. Introduction  Genomic D N A encodes for RNAs and proteins that are involved in nearly all cellular processes. Mutational changes may be induced by D N A damaging agents (chemicals and radiations), or arise as spontaneous reactions or replication errors. On the one hand, mutations are crucial for the survival of a species because they introduce genetic variations for natural selection and drive evolution. On the other hand, mutations generally have deleterious effects on cellular functions, especially when they result in abnormal RNAs and proteins or alter expression levels of gene products. Therefore, the cell has mechanisms to promote stability of the genome: first, the D N A replication machinery has inherent proofreading activity; second, several pathways exist to repair different types of D N A damage. D N A replication and repair are interconnected processes. First, D N A lesions may persist into S phase and block replication fork progression. In order for replication to restart, the lesions have to be either removed by repair mechanisms, or bypassed i f the cell is incapable of repairing the lesions (McGlynn and Lloyd, 2002). Second, the proofreading activity of the replication machinery is unable to remove some errors that occur during D N A replication. These errors are usually mismatches and small insertions or deletions, which can be dealt with by the mismatch repair pathway (Hoeijmakers, 2001). One feature of eukaryotic genomes is the abundance of simple tandem repeat sequences which occur at both chromosome ends and internal locations. Those that occur at chromosome ends are assembled into structures called telomeres. The presence of telomeres is crucial for genomic stability because they allow the cell to distinguish between natural chromosome ends and broken chromosomes. Failure to do so leads to chromosomal instability as a result of the cell's attempt to "repair" the ends. Simple tandem repeat sequences that occur at internal locations are called microsatellites - so called because during conventional D N A isolation methods, which result in mechanically sheared genomic D N A , such repeat sequences are separated from the bulk D N A upon density gradient centrifugation as satellite bands due to their different base compositions. Microsatellites usually have a repeat size of between one and six nucleotides, and span from a few up to > 100 nt in tract  1  length. The enrichment of microsatellites in a genome can be measured by the ratio of the frequency of observed microsatellites to the frequency predicted from random association of nucleotides. Genomic instability, or mutator phenotype, refers to an abnormal cell state associated with an increased rate of genomic alternations. It occurs when one or more mechanisms that maintain genomic integrity in the cell are defective. These mechanisms include mismatch repair (MMR), nucleotide excision repair (NER), base excision repair (BER), doublestranded break repair (DSB), as well as cell cycle checkpoints (Hoeijmakers, 2001; Kastan and Bartek, 2004). One form of genomic instability involves microsatellites (microsatellite instability), and is characterized by frequent changes in tract lengths. Genomic instability may also arise when defects in telomeres destabilize chromosomes. The following sections will provide background information on the maintenance of microsatellites and telomeres in the genome, with emphasis on the subject of my research: the role of a novel helicase in genomic stability.  1.1 Maintenance of repeat sequences 1.1.1 Distribution of microsatellites in eukaryotic genomes The distribution of microsatellites has been investigated in many species because of their role in genome evolution. Comparison of distribution of microsatellites in different genomes has revealed some general characteristics of these repeats: first, enrichment of microsatellite repeats increases with tract length for all six classes: mono-, di-, tri-, terra-, penta-, and hexanucleotides (Dieringer and Schlotterer, 2003; Metzgar et al., 2000). Second, the rate of increase in enrichment as a function of length is significantly lower in coding regions than in non-coding regions for nontriplet repeats (i.e. mono-, di-, terra-, and penta-), leading to a distribution where long nontriplet repeats are dramatically more abundant in non-coding regions than in non-coding regions. On the other hand, triplet repeats (i.e. tri- and hexanucleotides) are more evenly distributed between coding and non-coding regions (Field and Wills, 1998; Metzgar et al., 2000; Toth et al., 2000). This difference in distribution between nontriplet and triplet microsatellites was postulated to be the result of mutation bias, since length changes in triplet microsatellites are in general less deleterious than length  2  changes in nontriplet microsatellites, which would lead to frameshift mutations (Metzgar et al., 2000). Third, (A/T)n is much more abundant than (G/C)n except in Caenorhabditis elegans (Denver et al., 2004a; Toth et al., 2000). Fourth, (CG)n is the least abundant dinucleotides and is also extremely rare in all eukaryotic genomes analyzed (Toth et al, 2000). This rarity of (CG)n is likely to be attributed to methylation of cytosine in CpG and subsequent deamination of 5-methylcytosine to thymine (Schorderet and Gartler, 1992). Fifth, among the 10 trinucleotides, (ACG)n and (ACT)n are usually the least abundant (Toth et al, 2000). Mononucleotides are one of the most abundant microsatellite repeat types in almost all genomes analyzed. The two types of mononucleotides, (A/T)n and (G/C)n, have fairly different characteristics and properties (in this thesis, a slash (/) refers to complementary base pairs; for example, (G/C) refers to a string of 20 G's based paired to a string of 20C's, while 20  (G) refers to a just a string of G's). Crystallographic data have shown that (A/T)n sequences 20  have a rigid structure stabilized by additional non-Watson-Crick cross-strand hydrogen bonding (Nelson et al., 1987), whereas (G/C)n could form secondary structures such as triplexes and G-quadruplexes (see sections 1.1.5 and 1.1.6). It has been demonstrated that (G/C)n has a higher mutation rate than (A/T)n in both yeast and C. elegans (Denver et al., 2004a; Gragg et al., 2002). A higher mutation rate in (G/C)n is likely to occur in most organisms, resulting in the higher abundance of (A/T)n than (G/C)n found in all genomes analyzed (Dechering et al., 1998; Toth et al., 2000). A related phenomenon is that an increase in over-representation with increasing tract length is observed in (A/T)n but not in (G/C)n in various organisms, an exception being C. elegans (Dechering et al., 1998; Denver et al., 2004a). 1.1.2 Microsatellite instability While most microsatellites could be neutral sequences that accumulate in the genome due to errors in replication or recombination, numerous studies have provided strong evidence that some of these repeats are involved in gene expression and function (reviewed in Kashi et al, 1997). Direct evidence that microsatellites close to or within genes could affect gene expression was obtained from deletion studies, oligonucleotide competition assays, and mammalian gene reporter systems (Hamada et al., 1984; Lue et al., 1989; Meloni et al., 1998;  3  Struhl, 1985). The existence of polyglutamine and polyproline tracts in different organisms suggests that these tracts play a role in normal protein function (Holt and Koffer, 2001; Kashi et al., 1997). Expansion of trinucleotide repeats is the cause of a number of human diseases (reviewed in Cummings and Zoghbi, 2000). Expansion of trinucleotide repeats located within non-coding regions could affect gene expression in different ways. For instance, in Fragile X syndrome, expansion of the polymorphic (CCG)n repeat in the 5' untranslated region of the fragile X mental retardation gene (FMR1) results in hypermethylation of the repeat and the nearby promoter region and the subsequent inhibition of FMR1 transcription (reviewed in Cummings and Zoghbi, 2000). In Friedreich's Ataxia, the expanded (GAA)n repeat located in intron 1 of X25 forms triplex D N A structure causing gene silencing (Sakamoto et al., 1999). When located in coding regions, expansion of trinucleotide repeats becomes pathogenic when the encoded peptides are polyglutamine, an example being Huntington Disease: the expanded polyglutamine tracts cause protein aggregation that is toxic to neurons and lead to progressive neuronal dysfunction (Cummings and Zoghbi, 2000). Besides trinucleotide repeat expansion, another type of microsatellite instability related to human disease involves small insertions and deletions within microsatellites (Ionov et al., 1993; Zhang et al., 2001). Microsatellite instability could promote tumorigenesis by increasing the probability of mutations in genes involved in cell proliferation and survival. Numerous studies have associated frameshift mutations in certain genes, caused by length changes in mononucleotides within coding regions, and cancer of the gastrointestinal (GI) tract. Examples of these genes include IGFIIR (insulin-like growth factor II receptor) and BAXQ5CL2-associated X protein), which are involved in cell proliferation and cell death, respectively (Ouyang et al., 1997; Rampino et al., 1997). Defects in M M R cause the type of microsatellite instability seen commonly in heritable nonpolyposis colorectal cancer (HNPCC) and in some cases of sporadic colorectal cancer (Aaltonen et al., 1993; Liu et al., 1996; Peltomaki, 2001). M M R is the mechanism that deals with mismatches and small insertion or deletion loops that occur during D N A replication. In eukaryotes, the repair process starts with recognition of errors by complexes of the MutSrelated proteins, MSHs (MutS Homologs): the MSH2/MSH6 heterodimer is responsible for the repair of mismatches and single base insertion/deletion loops, whereas the MSH2/MSH3 heterodimer is responsible for single base and larger insertion/deletion loops (Kolodner and  4  Marsischky, 1999; Sia et al., 1997). MSHs then recruit M L H s (MutL homologs), which have been shown to form three different heterodimers in yeast: MLH1/PMS1 (Post-Meiotic Segregation), M L H 1 / M L H 3 , and M L H 1 / M L H 2 , with the first one being the major M L H for post-replication repair (Flores-Rozas and Kolodner, 1998; Harfe et al., 2000; Prolla et al., 1994; Wang et al., 1999). Downstream events involve excision of the newly synthesized strand, which contains the replication error, and subsequent gap-filling synthesis. However, the precise mechanism and exact players of these processes have not been established (Schofield and Hsieh, 2003). Consistent with the observation that MSH2 and M L H 1 are the common components in MutS and MutL complexes respectively, mutations in MSH2 and MLH1 account for the majority of H N P C C and sporadic colorectal cancers caused by deficiency in M M R (reviewed in Peltomaki, 2001). 1.1.3 Mechanisms of changes in tract length in microsatellites Various studies have calculated the mutation rate of microsatellites to be several orders of magnitude higher than random D N A sequences (Ellegren, 2000).This inherent instability of microsatellites is manifested as frequent changes in tract lengths, resulting in the highly polymorphic nature of the repeats. Two major mechanisms have been proposed to generate the level of polymorphism in microsatellites: slipped strand mispairing and unequal crossover (Levinson and Gutman, 1987; Smith, 1976). In the first mechanism, the template and the nascent strands dissociate during D N A replication. The repetitive nature of the sequence allows misalignment when the two strands re-associate, forming a loop structure. If the loop escapes repairing, the newly synthesized strand will be deleted i f the loop occurs on the template strand, or expanded if the loop occurs on the nascent strand (Levinson and Gutman, 1987). Because mismatch repair is responsible for repairing loop structures, it is foreseeable that deficiencies in this D N A repair pathway could lead to microsatellite instability. In the second mechanism, misalignment between sister chromatids or homologous chromosomes at the repeats followed by cross-over could result in changes in repeat number (Smith, 1976).  5  1.1.4 Parameters affecting stability of microsatellites Stability of microsatellites is affected by several parameters: base composition of the repeat, presence of variant repeats, length of microsatellite sequence, and orientation of the microsatellite with respect to replication origin. Mutation rates for (G/C)n and (A/T)n of the same lengths have been measured in yeast and mammalian cell culture. In both systems, (G/C)n was found to be more frequently mutated than (A/T)n, and this difference could be related to the scarcity of (G/C)n in eukaryotic genomes (Boyer et al., 2002; Harfe and Jinks-Robertson, 2000; Toth et al., 2000). Dinucleotide repeats that are self-complementary (CG and AT) are more unstable than the non-complementary repeats (GT and GA), indicating the formation of hairpin structures could be involved in the mechanism of instability (Bichara et al., 2000). In general, base composition of repeats influences the type of secondary structure that can potentially form as well as the stability of the structure, thereby affecting the mutation rate of the repeat. It has been observed that the presence of variant repeats correlates with normal alleles and their absence correlates with permutation alleles in both Fragile X syndrome and Spinocerebellar Ataxia type 1 (Chong et al., 1995b; Chung et al., 1993; Eichler et al., 1994). The presence of variant repeats within a microsatellite sequence has been directly shown to increase the stability of the repeat tract in E. coli and yeast (Bichara et al., 1995; Petes et al., 1997). The stabilization effect could be due to enhanced probability of correct realignment of complementary strands following dissociation during polymerase slippage, or decreased ability of the interrupted repeat tract to form secondary D N A structures (Petes et al., 1997). Numerous studies have demonstrated the dependence of microsatellite instability on length and orientation relative to replication origin of the repeat sequence. Because of the relevance of (CGG)n and (CTG)n trinucleotide repeats in human diseases, most studies examined these repeats specifically. Kang et al. (1995a) first showed that the orientation of (CTG)n affects its stability and frequency of deletion or insertion. Various lengths of (CTG)n were inserted in both directions into a plasmid with unidirectional replication origin: in orientation I, (CTG)n was the leading strand template; in orientation II, it was the lagging strand template (Figure 1-1). When the plasmids were transformed into E. coli, frequent deletions were observed when the insert was in orientation II, while the frequency of deletions was much less when it was in orientation I (Kang et al., 1995a). In both orientations, repeat instability increases with  6  length (Kang et al., 1995a). The authors excluded the possibility that deletions were transcription-dependent because the inserts were located in the non-transcribed region of the plasmid. Expansions were rare but could be detected by cloning (Kang et al., 1995a). Interestingly, the majority of the expansions were in orientation I. Hence, when (CTG)n is the template strand of lagging strand synthesis, deletions are more prominent than expansion; whereas when it is on the nascent strand of lagging strand synthesis, expansions are more frequent than deletions (Kang et al., 1995a). The length- and orientation-dependent instability is suggestive of a replication-related process that involves secondary D N A structures (Figure 1-1) (Kang et al., 1995a). Length- and orientation-dependent instability of (CTG)n was also observed in yeast (Freudenreich et al., 1998; Freudenreich et al., 1997; Maurer et al., 1996; Miret et al., 1997). For (CGG)n, instability is more pronounced when (CGG)n is the lagging strand template and when tract length increases (Balakumaran et al., 2000; Hirst and White, 1998; Shimizu et al., 1996; White et al., 1999). These data support a model in which (CGG)n and (CTG)n repeats form stable secondary structures on the lagging strand, leading to misalignment and hence deletion (if structure forms on the template strand) or expansion (if structure forms on the nascent strand). Besides trinucleotides, the dependence on the length of dinucleotides in repeat stability has also been documented in E. coli, yeast, and human (Levinson and Gutman, 1987; Wierdl et al., 1997; Yamada et al., 2002). 1.1.5 Secondary D N A structures formed by microsatellites The most stable conformation adopted by random D N A under physiological conditions is the right-handed B - D N A . Non-random D N A sequences, such as purine-rich tracts, alternating purine-pyrimidine segments, or inverted repeats, can adopt non-B type D N A conformations, such as Z - D N A , hairpins, cruciforms, triplexes, and quadruplexes (see reference below). The structures of different microsatellites have been characterized in vitro. Repeats with alternating purine and pyrimidine nucleotides (e.g. (AT)n and (GC)n) can form Z - D N A , which is a left-handed helix that is longer and thinner than B - D N A (Wells, 1988). Hairpins are formed by inverted repeats, but all three trinucleotide repeats that are associated with human diseases are also capable of forming these structures in vitro (Mitas, 1997). A triplex is the structure formed when a third strand binds the major groove of a helix in one of two conformations, depending on whether the third strand is homopyrimidine (parallel) or  7  homopurine (antiparallel). Triplexes are formed by purine»pyrimidine sequences (Gilbert and Feigon, 1999), the (GAA/TTC)n trinucleotide repeat (Ohshima et al., 1996), and the (TC/GA)n dinucleotides repeat (Hanvey et al., 1988; Johnston, 1988). G-quadruplex, formed by G-rich D N A , is essentially a stack of G-quartets, which are planar structures in which four guanine residues interact by Hoogsteen base-pairing (Figure 1-2A). The G-quadruplex structure was first proposed by Gellert et al. (1962), based on optical and X-ray diffraction studies of concentrated solutions of G M P (Guanosine 5' monophosphate) (Gellert et al., 1962). A G - quadruplex can be in one of many conformations: one, two or four strands may  Orientation II  Deletion  Figure 1-1. Model proposed by Kang et al. (1995) to explain orientation-dependent expansions and deletions of (CTG)n in E. coli. (Figure adapted from Kang et al., 1995).  8  be involved and these strands may be in parallel or anti-parallel orientations (reviewe in Shafer and Smirmov, 2000) (Figure 1-2B). G-quadruplex is different from other secondary D N A structures in that its formation requires the presence of metal cations, which coordinate along the central axis of the structure. A fairly wide variety of cations can induce G quadruplex formation, their ionic radius partly determines how effective they are in stabilizing the structure (reviewed in Shafer and Smirnov, 2000). However, it is likely that only K , N a , C a +  +  2+  and M g  2 +  are relevant in vivo. The concentrations of these ions in the  nucleus could influence which conformation of a G-quadruplex is adopted by G-rich D N A (reviewed in Shafer and Smirnov, 2000). The only trinucleotide that has been documented to form a G-quadruplex in vitro is (CGG)n (Fry and Loeb, 1994). It is expected that (G)n would form a G-quadruplex readily. However, (Sundquist and Klug, 1989) did not observe any inter- or intra- strand association when telomere-like oligonucleotides containing a (G)  12  overhang were separated on a native gel after incubation in the presence of N a . On the other +  hand, based on the pattern of protection from methylation by dimethyl sulfate (DMS), (Panyutin et al., 1990) deduced that both (G) and (G) formed G-quadruplexes. 27  37  Figure 1-2. The G-quadruplex structure. Shown here are (A) the structure of a G-quartet and (B) some conformations of a G-quadruplex. (Figure adapted from Shafer and Smirnov, 2000)  9  It has been documented that microsatellites that are capable of forming secondary structures cause replication blockage. During in vitro D N A synthesis from long (TCVGA)n templates, it was observed that replication was blocked around the middle of the repeat tracts (Baran et al., 1991; Lapidot et al., 1989). Baran et al. (1991) showed that the block in replication was eliminated when the nucleotide analogues 7-deaza dATP and 7-deaza dGTP, in which the N7 atom in adenine and guanine is replaced by a carbon atom, were used in D N A synthesis reactions. Because the model of (TC/GA)n forming triplexes requires that N7 be available for Hoogsteen hydrogen bonding, the results from using nucleotide analogues are suggestive of D N A secondary structures being the block of D N A replication (Baran et al., 1991). This hypothesis was further supported by the fact that inclusion of single-strand binding protein also eliminated the arrest in D N A synthesis, and that the arrest was dependent on pH, because triplex formation by (TC/GA)n requires protonation of the cytosine residues (Baran et al., 1991). Similar studies led to the same conclusion that formation of secondary structures may be responsible for replication arrest in (CTG)n and (CGG)n trinucleotide repeats (Kang et al., 1995b; Usdin and Woodford, 1995). Later it was shown that long (CTG)n and (CGG)n indeed block replication fork progression in vivo (Samadashwily et al., 1997). Using (CTG)n and (CGG)n of various lengths cloned into a plasmid that replicates unidirectionally, Samadashwily et al. (1997) studied replication fork movement through the repeats in E. coli by two-dimensional gel electrophoresis and demonstrated replication blockage within the repeats in a length- and orientation-dependent manner. The authors ruled out the possibility of protein-binding or transcription at the repeats causing replication blockage, and thus arrived at the conclusion that D N A secondary structure formation is the most likely cause of blockage. Replication blockage may be responsible for polymerase slippage at microsatellites, leading to frequent changes in tract length (Kang et al., 1995a). 1.1.6 G-quadruplex 1.1.6.1 Potential involvement of G-quadruplexes in biological processes The G-quadruplex structure has received much attention because of its potential involvement in different biological processes. One of the first proposals for the functional roles of Gquadruplex was put forth by Sen and Gilbert (1988), who speculated that G-rich sequences  10  along the chromosome could self-associate to bring the four chromatids together during meiosis, based on their observation that G-rich motifs from the immunoglobulin heavy chain switch regions self-associate in physiological salt concentrations. Since then, various studies have provided evidence for G-quadruplex having biological functions. Once formed, G-quadruplexes are very stable structures that are not easily disrupted. Sen and Gilbert (1988), upon the discovery that naturally occurring G-rich sequences are capable of forming G-quadruplex in vitro, speculated that the cell may control G-quadruplex formation by proteins that bind or melt the structure. Indeed, a number of such proteins have been identified. Two of the five identified human RecQ helicases, B L M and W R N , whose mutations cause Bloom Syndrome and Werner Syndrome respectively, and the only RecQ homolog in S. cerevisiae, Sgslp (slow growth suppressor), are all able to unwind G quadruplexes in vitro (Fry and Loeb, 1999; Sun et al., 1999; Sun et al., 1998). Significantly, a G-quadruplex is the preferred substrate over duplex D N A for all three helicases (Fry and Loeb, 1999; Sun et al., 1999; Sun et al., 1998). While both B L M and Sgslp can unwind Gquadruplexes formed by telomeric sequences having a 3' tail (Sun et al., 1998; Sun et al., 1999), W R N is only able to unwind those formed by (CGG)n with either a 5' or 3' tail (Fry and Loeb, 1999). This difference in substrate specificity was postulated to be relevant to the distinctive phenotypes in Werner Syndromes and Bloom Syndromes (Fry and Loeb, 1999). Another helicase, SV40 large-T antigen, has also been shown to unwind G-quadruplex formed by different sequences, including the immunoglobulin switch region and Tetrahymena telomeric repeats (Baran et al., 1997). 1.1.6.2 Implications of the G-quadruplex structure on gene expression One possible functional role of G-quadruplex is control of gene expression. The best studied example is c-MYC, which is an oncogene that affects cell proliferation, differentiation and apoptosis, and whose overexpression has been detected in many cancers (reviewed in Pelengaris et al., 2000). A high percentage of c-MYC transcription is controlled by a G-rich 27 bp nuclease hypersensitivity element (NHE III,) found upstream of the PI promoter, which is one of four promoters that control transcriptional expression of c-MYC (reviewed in Siddiqui-Jain et al., 2002). The ability of the purine-rich strand of the element to form a G quadruplex was investigated by D M S modification (Phan et al., 2004; Siddiqui-Jain et al., 2002; Simonsson et al., 1998) and later confirmed by N M R studies (Ambrus et al., 2005; 11  Phan et al., 2004). Using the luciferase reporter assay, it was demonstrated that mutations that were expected to disrupt the proposed G-quadruplex structure led to a three-fold increase in gene expression (Siddiqui-Jain et al., 2002). Such results are compatible with a model in which formation of G-quadruplex by N H E III, silences expression of c-MYC. A G quadruplex is also believed to form in the insulin gene-linked polymorphic region (ILPR), located 363bp upstream of the human insulin gene (Catasti et al., 1996; Hammond-Kosack and Docherty, 1992). The proposed G-quadruplex structure has been hypothesized to affect transcription by binding to the transcription factor Pur-1 (Lew et al., 2000). 1.1.6.3 Implications of the G-quadruplex structure on recombination Other non-telomeric proteins have also been characterized to bind or display enzymatic activities on G-quadruplexes. The Maizels group studied LR1, a B cell-specific D N A binding factor that was postulated to play a role in regulating immunoglobulin class switch recombination, a process that depends on transcription of the G-rich switch (S) region found upstream of each constant (C) region (Snapper et al., 1997; Williams and Maizels, 1991). They later found that LR1 is composed of two subunits, nucleolin and heterogeneous nuclear ribonucleoprotein D (hnRNP D), both of which bind G-quadruplexes with high affinity in gel mobility shift assays (Dempsey et al., 1998; Dempsey et al., 1999; Hanakahi and Maizels, 2000; Hanakahi et al., 1999). Dempsey et al. (1999) proposed that the G-rich non-template strand of S regions could form G-quadruplexes during transcription. If the structure is formed on both donor and acceptor S regions, interaction with LR1 could juxtapose these two regions for recombination. Further evidence that supports the involvement of G-quadruplex in immunoglobulin class switch recombination is the identification of a nuclease specific for G-quadruplex, G Quartet Nuclease 1 (GQN1), whose levels are elevated in a transformed B cell line relative to HeLa cell line (Sun et al., 2001). Recently, Duquette et al. (2004) provided direct evidence for the existence of G-quadruplex in living cells. Using an in vitro transcription system, in which G-rich sequences from mammalian immunoglobulin S regions or human telomeric repeats were placed downstream of a T7 promoter, the authors observed loop structures where the transcribed strand was a D N A / R N A hybrid and the non-transcribed G-rich strand contained a G-quadruplex (Duquette et al., 2004; Figure 1-3). G-quadruplex formation was demonstrated by GQN1 cleavage, binding to a recombinant truncated derivative of nucleolin, and D M S methylation protection assay (Duquette et al., 2004). Such  12  loop structures, which were termed G-loops, were not observed when transcription did not occur or when the G-rich strand was on the transcribed strand (Duquette et al., 2004). Existence of G-loops in living cells was demonstrated in E. coli that lacks RNase H and RecQ, both of which were expected to destabilize the structure (Duquette et al., 2004). The observation that a G-quadruplex could form during transcription of the S regions provides addition support for the model proposed by Dempsey et al. (1999), in which interaction of Gquadruplexes with LR1 could bring the donor and acceptor S regions together for recombination. 1.1.6.4 Evidence of G-quadruplex formation at telomeres Telomeric repeats are typically G-rich (see section 1.3). Williamson et al., (1989) provided evidence for formation of G-quadruplex by telomeric sequences from Oxytricha and Tetrahymena: in the presence of Na , the oligonucleotides had a higher mobility on a native +  gel; certain guanine residues of the oligonucleotides were protected from methylation at N7 by D M S in the presence of Na ; in addition, irradiation by U V caused intra-molecular cross+  linking between two thymine residues that were 2 repeats away, indicative of some fold-back structures (Williamson et al., 1989). Sundquist and Klug (1989) also deduced the generation of G-quadruplex by dimerization of hairpins formed by oligonucleotides of the Tetrahymena telomeric sequence (Sundquist and Klug, 1989). Adoption of the G-quadruplex structure by telomeric sequences was later confirmed by N M R studies and crystallography (Kang et al., 1992; Parkinson et a l , 2002; Smith and Feigon, 1992; Wang and Patel, 1993) Some telomeric proteins have been shown to interact with G-quadruplexes, supporting the notion that telomeric D N A forms G-quadruplex in vivo. In Oxytricha nova, a heterodimeric protein consisting of a and p subunits forms the "telosome" at the singlestranded  T4G4T4G4  overhang of macronuclear telomeres (Gottschling and Cech, 1984;  Gottschling and Zakian, 1986; Price and Cech, 1987). It has been shown that on its own, the P subunit promotes association of telomeric D N A by G-quadruplex formation (Fang and Cech, 1993a). The region of the P subunit responsible for this structure-promoting activity is separable from the region that interacts with the a subunit and D N A to form the telosome, implying that the p subunit participates in two different activities at the telomere (Fang and Cech, 1993a; Fang and Cech, 1993b). Another telomeric protein that has been shown to  13  interact with G-quadruplexes is Raplp (repressor/activator protein 1), which is a sequencespecific double-stranded telomeric DNA-binding protein and affects telomere length regulation in S. cerevisiae (Conrad et al., 1990; Giraldo and Rhodes, 1994; Lustig et al., 1990). Giraldo and Rhodes (1994) demonstrated that in the presence of Raplp, an oligonucleotide containing four overlapping Raplp-binding telomeric sequences adopts the G-quadruplex structure as evident by D M S methylation protection. Since the oligonucleotide is susceptible to D M S methylation in the absence of Raplp under the particular assay conditions, the results suggest that Raplp may promote formation of G-quadruplex by yeast telomeric D N A (Giraldo and Rhodes, 1994). Besides those mentioned above, many other proteins have been identified to interact with G-quadruplexes, suggesting that these structures may be involved in various biological processes (reviewed in Shafer and Smirnov, 2000).  1.2 Helicases and genome maintenance 1.2.1 Structure and classification of helicases As mentioned above, G-quadruplex can be unwound by certain helicases in vitro. Helicases are enzymes that utilize the energy derived from hydrolysis of nucleoside 5'-triphosphate to separate two complementary nucleic acid strands. Unwinding of the two D N A strands is prerequisite to many cellular processes involving nucleic acids, including D N A replication, D N A repair, recombination, and transcription. Because of this fundamental role of helicases in D N A metabolism, they are found in all organisms. A l l helicases share several common biochemical properties, including nucleic acid binding, NTP binding and hydrolysis, and NTP hydrolysis-dependent unwinding of duplex nucleic acids. They can be categorized according to their biochemical activities into either D N A or R N A helicases, and either 5 ' - > 3 ' o r 3 ' — » 5 ' , according to the directionality of their movement with respect to the strand they initially bind and move along (reviewed in Tuteja and Tuteja, 2004). Based on amino acid sequence analysis from a diverse collection of organisms, helicases (and putative helicases) have been classified into four groups: superfamily 1 (SF1), superfamily 2 (SF2), superfamily 3 (SF3) and family 4 (F4) (Gorbalenya and Koonin, 1993; Hall and Matson, 1999) (Figure 1-3). A l l helicases carry two sequence signatures, Walker A and Walker B motifs (also called motif I and motif II  14  Superfamily 1 J++xGxAGoGRSL * A P Tf  la JXX+XXXOOL  i t t - f - D E x o j.  III  IV  III  IV  i+t+tGOxod'^; xx+xooxRL  V  VI  V  VI  Jxxr+xxxQG+0+OOVi *t S K  Superfamily 2 I  la  II  |++xxxoGxGKT^™Jx++ + x P o o ^ « • • • D E x H ™ « j + x * - S A T x x x ^ ~ + + F x x o x o _ g + x T x x x x x G + o + x O + g M QxxGRxxR• : _ . i \ J \ .T.G.S. < :„_Y__ ...J L §_ „.i i i  Superfamily 3 A i  ..C _  B a  Family 4 1  i  !„ ,.,,SSJ u  Ss F  1a  x + x + ^ x A R x x + G R T ^ — V L x * S L O ^ ^ + I + » D Y V ^ ^ I x x I x x o l K A + A x o L x P + x x + x Q ^ ^ J PxxxDLRxSGxIxQxADxH SS JSr^_fi_„BM_B~ „„.Hlf^ it BS. JL.X ^ f ^ ^ v _ , i j;  Figure 1-3. Classification of helicases. The classification was proposed by Gorbalenya and Koonin (1993) and Hall and Matson (1999). SF1 and SF2 are characterized by seven helicase motifs as depicted by the boxes. Letters within the boxes are conserved residues. For SF3, upper case letters indicate presence in more than 50% of helicases in this superfamily; lower case letters indicate presence in more than 50% of the D N A or R N A viral helicases that belong to this superfamily. F4 helicases carry five conserved motifs, and letters within the boxes are conserved residues. "+" refers to hydrophobic residues, "o" refers to hydrophilic residues, and " x " represents residues that are not restricted to being hydrophobic or hydrophilic. (Figure adapted from Hall and Matson, 1999)  respectively in SF1, SF2, and F4 helicases), which are involved in NTP-binding (Gorbalenya and Koonin, 1993; Hall and Matson, 1999). SF1 and SF2 are structurally very similar helicases that might have evolved from a common ancestor (Gorbalenya and Koonin, 1993). Helicases in these two superfamilies have seven conserved motifs (I, la, II, III, IV, V , VI) with similar sequences, arrangements, and predicted secondary structures (Figure 1-3) (Gorbalenya and Koonin, 1993; Hall and Matson, 1999). Crystallography showed that the seven motifs are closely associated in the tertiary  15  structure of the helicase, forming a pocket that binds nucleotides (Korolev et al., 1997; Subramanya et al., 1996). Therefore, all seven motifs may be involved in certain aspects of A T P binding/hydrolysis (Korolev et al., 1997; Subramanya et al., 1996). Within motif II, an aspartic acid residue and the following glutamic acid residue (DE) are completely conserved, and mutational studies have established that they are critical for A T P hydrolysis but not A T P binding (Brosh and Matson, 1995; Pause and Sonenberg, 1992). SF1 and SF2 encompass both D N A and R N A helicases from archaea, eubacteria, prokaryotes and eukaryotes (Gorbalenya and Koonin, 1993; Hall and Matson, 1999). Examples of SF1 helicases include E. coli UvrD (also called helicase II), which is involved in D N A repair (Lahue et al., 1989), and yeast Upflp, which is involved in nonsense-mediated mRNA decay (Leeds et al., 1991). SF2 comprises the largest group of helicases including the DEAD-box R N A helicases and other D N A helicases with the conserved D E x H box within motif II, such as the RecQ helicases (Gorbalenya and Koonin, 1993; Hall and Matson, 1999). SF3 helicases contain only three conserved motifs, namely A , B , and C. Motifs A and B carry the Walker A and Walker B sequences, respectively (Gorbalenya and Koonin, 1993; Hall and Matson, 1999). A n example of SF3 helicase is the viral SV40 T antigen, which is involved in D N A replication (Goetz et al., 1988). F4 helicases have five conserved motifs (I, la, II, III, IV) and include the DnaB-related proteins involved in D N A replication (Gorbalenya and Koonin, 1993; Hall and Matson, 1999). For example, bacteriophage T7 gp4 protein is required for D N A replication (Richardson, 1983), and E. coli DnaB is the replication fork helicase that unwinds parental D N A during replication (LeBowitz and McMacken, 1986). The fundamental role of helicases in nucleic acid metabolism suggests that deficiencies in helicases could have severe consequences. In human, mutations in five helicases are known to cause diseases: X P B (xeroderma pigmentosum complementation group B), X P D (xeroderma pigmentosum complementation group D), B L M (Bloom syndrome), W R N (Werner syndrome), and RECQL4 (RecQ-like 4). Mutations in another helicase, BACH1/BRIP1 (Brcal-Associated C-terminal Helicase 1 / BRCA1-interacting protein 1), are believed to result in cancer predisposition.  16  1.2.2 X P B a n d X P D Defects in X P B or X P D of SF2 (Figure 1-3) could result in xeroderma pigmentosum (XP), Cockane syndrome (CS), or Trichothiodystrophy (TTD), which are clinically distinctive disorders (reviewed in Lehmann, 2001). X P B and X P D are two of the nine subunits of the transcription factor I I H (TFIIH) (Schaeffer et al., 1994; Schaeffer et al., 1993). The two helicases are thought to unwind sequences at promoter regions during transcription initiation, and at certain D N A damage sites (e.g. UV-induced lesions) that are repaired by the nucleotide excision repair (NER) pathway (Evans et al., 1997; Holstege et al., 1996). The helicase activity of X P B appears to be required for transcription, because mutations in the ATP-binding site of yeast Rad25p (homolog of XPB) result in defects in both transcription and N E R (Park et al., 1992; Sung et al., 1988). In contrast, mutations in the ATP-binding site of Rad3p (homolog of XPD) only affect N E R (Park et al., 1992; Sung et al., 1988). Furthermore, in vitro transcription assays using a purified K48R X P D mutant protein (which is mutated within the conserved Walker A sequence) showed that the helicase activity of X P D is dispensable for transcription (Winkler et al., 2000). Results from these and other studies have led to the conclusion that X P B helicase activity is essential for both transcription and repair functions, while X P D may contribute to TFIIH functioning in transcription by some other type of activity, such as stability of the transcription initiation complex (reviewed in Lehmann, 2001). 1.2.3 RecQ B L M , W R N and RECQL4 belong to the family of RecQ helicases, which is a subgroup within SF2 (Figure 1-3) (Gorbalenya and Koonin, 1993). Mutations in these proteins result in Bloom syndrome (BS), Werner syndrome (WS) and Rothmund-Thomson syndrome (RTS) respectively. A l l these syndromes are characterized by predisposition to cancer (reviewed in Mohaghegh and Hickson, 2001). WS is also well-known for its premature-aging phenotype. BS cells show a high frequency of sister chromatid exchanges (SCEs) and illegitimate recombination (reviewed in Mohaghegh and Hickson, 2001). WS cells exhibit increased sensitivity to certain chemicals (e.g. 4-nitroquinoline 1-oxide, 4NQO), elevated frequencies of large deletions and translocation, and dramatically reduced replicative life-span in culture (reviewed in Bachrati and Hickson, 2003). RTS cells are characterized by a high incidence of  17  aneuploidy (Lindor et al., 2000). Two other human helicases, R E C Q L and RECQL5, also belong to this family, but no disease has been associated with them (Khakhar et al., 2003; Kitaoetal., 1998). Among the five human RecQ homologs, B L M and W R N have been studied the most extensively. Both are 3' -> 5' helicases that unwind G-quadruplexes, bubbles, 4-way junctions (models of the Holliday junction recombination intermediate) efficiently but not the typical helicase substrates of duplex oligonucleotides with 3' or 5' overhangs (Constantinou et al., 2000; Gray et al., 1997; Karow et al., 1997; Karow et al., 2000; Mohaghegh et al., 2001; Sun et al., 1998). W R N differs from B L M in that it has a C-terminal exonuclease domain and displays exonuclease activity in addition to its helicase activity (Huang et al., 1998). W R N has been implicated in D N A replication: it co-purifies with the D N A replication complex on a sucrose gradient (Lebel et al., 1999) and interacts with several proteins involved in D N A replication, including P C N A (proliferating cell nuclear antigen), FEN1 (flap endonuclease 1) and R P A (replication protein A) (Bachrati and Hickson, 2003; Brosh et al., 1999; Brosh et al., 2001; Lebel et al., 1999). W R N also interacts physically and functionally with proteins involved in genomic stability, such as p53 and K u proteins (Bachrati and Hickson, 2003; Blander et a l , 1999; Cooper et al., 2000). A role for W R N at the telomere is also evident. WS fibroblasts display accelerated rates of telomere length in culture (Schulz et al., 1996), and expression of hTERT in these cells extends cellular lifespan and partially rescues their sensitivity to 4NQO (Hisama et al., 2000; Wyllie et al., 2000). Furthermore, W R N has been shown to colocalize with the telomeric repeat binding proteins TRF1 and TRF2 by indirect immunofluorescence, and associate with telomeric D N A by chromatin immunoprecipitation in A L T cells (cells that maintain telomeres in a telomeraseindependent manner, presumably by homologous recombination) (Johnson et al., 2001; Opresko et al., 2004; Opresko et al., 2002). In vitro studies showed that TRF2 interacts with W R N and stimulates the helicase activity of W R N (Opresko et al., 2002). Recently, it was demonstrated that a telomeric D-loop substrate (where 3' overhang invades the duplex region of the telomere; see section 1.3.3) can be dissociated by W R N , which cleaves the invading strand (INV) and releases the cleaved INV (Opresko et al., 2004). This cleavage and release of INV is dependent on both the helicases and the exonuclease activities of W R N (Opresko  18  et al., 2004). Moreover, TRF1, TRF2 and R P A are capable of influencing the relative abundance of different cleavage products (Opresko et al., 2004). The high frequency of SCEs in BS cells suggests that B L M may function to suppress illegitimate recombination. A role of B L M in homologous recombination is supported by the following observations: (1) B L M interacts with RAD51, which is a crucial component in homologous recombination (Wu et al., 2001); (2) the increase in SCEs associated with B L M deficiency is diminished in a RAD54-/- background in chicken DT40 cell line (Wang et al., 2000a); (3) B L M promotes branch migration of Holliday junction, which is a recombination intermediate (Karow et al., 2000); (4) B L M resolves a double Holliday junction substrate, the intermediate formed when both ends of a double-stranded break invade the homologous sequence (Wu and Hickson, 2003). It does this in a hTOPOIIIa-dependent manner (Wu and Hickson, 2003). B L M has also been shown to interact with R F A and FEN1, which are involved in D N A replication (Brosh et al., 2000; Sharma et al., 2004). In addition, coimmunoprecipitation and gel filtration revealed that B L M exists as a complex with many proteins that participate in maintaining genomic stability, including components of the mismatch repair pathway, A T M , RAD50, and BRCA1 (Wang et al., 2000b). B L M has also been implicated in telomere metabolism. Both TRF1 and TRF2 colocalize with B L M in A L T cells and coimmunoprecipitate with B L M in vitro (Lillard-Wetherell et al., 2004; Stavropoulos et al., 2002). Furthermore, both proteins affect the unwinding activity of B L M (Lillard-Wetherell et al., 2004; Opresko et al., 2002). 1.2.4 BACH1/BRIP1 BACH1/BRIP1, which also belongs to SF2 but differs from the RecQ families based on amino acid sequence comparison (Menichini and Linial, 2001), was identified in a screen for proteins that interact with the B R C T (BRCA1 C-terminal) domain of B R C A 1 (breast cancer 1) (Cantor et al., 2001). Mutations in BRCA1 occur in ~50% of families with inherited breast cancer, and ~80% with combined breast-ovarian cancer (Ford et al., 1998). The precise function of BRCA1 is unclear, but it has been implicated in a multitude of cellular processes, and its role in D N A repair has received the most attention. BRCA1 participates in the sensing and signaling of D N A lesions and cell cycle checkpoint control; at the same time, it is associated with downstream effector molecules in D N A repair (reviewed in Starita and  19  Parvin, 2003; Venkitaraman, 2002). Therefore, BRCA1 has been proposed to be a link between D N A damage sensing and biological responses (Starita and Parvin, 2003; Venkitaraman, 2002). BACH1/BRIP1 is a 5' ->• 3' helicase that is capable of unwinding both D N A r D N A and D N A : R N A substrates (Cantor et al., 2004). Its N-terminal 880 residues contain the seven signature motifs of a D E A H helicase of SF2, as well as a nuclear localization signal (NLS) (Cantor et al., 2001). Its C-terminal domain is required for interaction with B R C A 1 (Cantor et al., 2001). Interaction with the B R C T domain of B R C A 1 is dependent on phosphorylation of Ser990 in the C-terminal domain of BACH1/BRIP1 (Rodriguez et al., 2003; Y u et al., 2003). Both phosphorylation at Ser990 of BACH1/BRIP1 and coimmunoprecipitation between BACH1/BRIP1 and BRCA1 only occur in S to G2/M but not in G l (Yu et al., 2003) . This cell cycle-regulated interaction is consistent with colocalization studies by Cantor et al. (2001). Cells in which BACH1/BRIP1 expression has been inhibited by R N A i fail to arrest at G2/M after gamma irradiation, suggesting that BACH1/BRIP1, like B R C A 1 , is involved in cell cycle checkpoint control (Yu et al., 2003; Rodriguez et al., 2003). Screening of 65 individuals with early onset breast cancer carrying no BRCA1 or B R C A 2 mutations revealed two cases of heterozygous missense mutations that occurred within the helicase domain of BACH1 (Cantor et al., 2001). Neither of the two mutations was detected in control individuals, arguing against their being polymorphisms (Cantor et al., 2001).  1.3 Repeat sequences at chromosome ends As mentioned in section 1.1, the eukaryotic genome is interspersed with simple repeat sequences. One chromosomal location where the presence of simple repeats is known to be crucial for survival of the cell is the end of the chromosome. Chromosome ends exist as a structure called telomeres, which are composed of short tandem telomeric repeats and associated proteins (reviewed in Blackburn, 2001). Telomeres have at least three functions. A l l eukaryotic cells encounter the end replication problem, which refers to the inability of the conventional replication machinery to fully replicate the ends of linear chromosomes (reviewed in Smogorzewska and de Lange, 2004) . This is due to the fact that D N A can only be synthesized from 5' to 3', so after the  20  R N A primer at the 3' end is removed, a gap that cannot be filled remains. In the next round of D N A replication, the gap would be converted into a shortened end (Figure 1-4). This loss of genetic materials is known as replicative loss. The presence of telomeric repeats therefore  •3' •5' Replication ,  .3'  3' — 5*  lagging strand telomere leading strand telomere  fiNAprimtftemovai and gap filling  |  •3' •5"  _  _ "  3' overhang  blunt end  3  5"  degradation of 5' ends -generates long 3' overhangs •3'  5  r  .  * 3' 5*  Figure 1-4. D N A replication is predicted to generate asymmetric chromosome ends. After removal of R N A primers, the strand replicated by lagging strand synthesis (lagging strand telomere) is left with a 3' overhang corresponding to the size of the primer. The strand replicated by leading strand synthesis (leading strand telomere) is blunt ended because replication can proceed to the very end. However, the presence of long overhangs in some organisms and the requirement of a 3' overhang to form a t-loop structure imply that some end processing occurs after D N A replication to generate overhangs of appropriate lengths on both ends. (Figure adapted from Makarov et al., 1997)  21  prevents replicative loss from affecting important coding sequences. A second function of telomere is to allow the differentiation between natural chromosome ends and doublestranded breaks (reviewed in Blackburn, 2001). Double-stranded breaks induce a D N A damage response, which includes arrest of cell cycle progression, attempts of D N A repair by homologous recombination (HR) or non-homologous end-joining (NHEJ), and apoptosis i f repair fails (Sancar et al., 2004). Mistaking chromosome ends as double-stranded breaks is disastrous because "repair" of chromosome ends would lead to chromosome fusions and subsequent chromosome breakage during cell division. Therefore, telomeres act as protective caps for chromosomes to prevent such fusions and breakage from occurring (Blackburn, 2001). Telomeres are also involved in the correct positioning of chromosomes in the nucleus. Telomere clustering (known as the bouquet structure) during early meiosis is believed to have a role in chromosome pairing and synapsis (Bass, 2003). In interphase, chromatids are anchored at the nuclear envelop (Taddei and Gasser, 2004). In yeast, such anchorage requires K u proteins (Laroche et al., 1998).  1.3.1 Telomeric D N A The first telomeric sequence was identified in Tetrahymena and was found to consist of T2G4 tandem repeats (Blackburn and Gall, 1978). Since then the telomeric sequence of many species has been characterized (Wellinger and Sen, 1997). In most of them, it is composed of short G-rich tandem repeats of different but related sequences (Table 1-1). Exceptions include some species of fungus and Drosophila. For instance, telomeres in S. cerevisiae are composed of irregular repeats of  TG1.3;  in Kluyveromyces, they are long 25bp tandem  repeats; in Drosophila, transposons instead of telomeric repeats are found at chromosome ends (McEachern and Blackburn, 1994; Shampay et al., 1984). The length of telomeric D N A varies widely among different species, from ~300bp in S. cerevisiae (Wright et al., 1992) to >100kb in Mus musculus (Kipling and Cooke, 1990; Starling et al., 1990). Telomere length also varies among different chromosomes in the same cell and among different cells from the same source (de Lange et al., 1990; Martens et al., 1998; Wicky et al., 1996; Zijlmans et al., 1997). Telomere length is a balance between telomere-shortening events (replicative loss and degradation/processing) and telomere-lengthening events (telomerase elongation and telomerase-independent extension) (Blackburn, 2001). Replicative loss originates from the  22  Table 1-1. Telomeric repeats in different organisms Organism  Telomeric repeat sequence  Protozoa Tetrahymena Glaucoma Oxytricha Euplotes Stylonychia Paramecium Trypanosoma Giardia  T G T G 2  4  2  4  T4G4 T4G4 T4G4  T [T7G]G T AG TAG, T [T/C]AGi 2  3  2  Plasmodium Theileria  3  2  T3-4AG,  Slime moulds Dictyostelium Physarum Didymium Fungi Neurospora Podospora Cladosporium Histoplasma Ctyptococcus Saccharomyces KJuyveromyces Candida albicans Candida tropicalis 4443 Candida guilkrmondii Schizosaccharomyces Pneomocystis Plants Chlamydomonas Chiorella Arabidopsis Zea mays  AG, . T AG T AG,  8  2  3  2  T T T T  2  2  2  2  AG AG AG AG  3  3  3  3  T AG3. (TG),. TG _3 ACG AT GAT AG TATGTG TGT ACG ATGTCTA CT CT G TGT A G ATGTCACGATCAT G TGT ACTG TGT 2  5  6  2  3  2  2  2  2  2  2  2  2  2  2  2  2  2  2  T j . A C A O - I CO- I G , _ 2  T AG 2  6  3  T4AG3 T3AG3 T3AG3 T3AG3  Hordeum (Barley) Nicotiana Lycopersicum (Tomato) Invertebrates Ascaris Parascaris Caenorhabdilis Bombyx (and other Lepidoptera) Vertebrates Homo sapiens Many other vertebrates  T3AG3 T3AG3  T (T/A)AG, 2  T AG C T GCA T AG C 2  2  2  2  2  T AG 2  2  T AG, T AG, 2  2  (adapted from Wellinger and Sen, 1997)  23  inability to replicate telomere ends completely, and the rate of this loss depends on the size of the R N A primer laid down by Pola of the D N A replication holoenzyme (Blackburn, 2001). The size of this primer should be similar in all eukaryotic cells because of the high degree of conservation in the D N A replication machinery (reviewed in Baker and Bell, 1998). However, the actual rate of telomere shortening in the absence of telomerase elongation varies widely, from a few nucleotides per cell division in S. cerevisiae to ~100bp per population doubling (PD) on average in mammalian cells in culture (Huffman et al., 2000; Lundblad and Szostak, 1989). Therefore, the relatively high rate of telomere loss in some species suggests that there is active processing at chromosome ends. Telomerase-dependent elongation is the major mechanism to counteract telomeric D N A loss in most normal cells (reviewed in Blackburn, 2001; Smogorzewska and de Lange, 2004). In yeast without functional telomerase, rare survivors arise that are able to use recombination to maintain telomeres (Lundblad and Blackburn, 1993; Nakamura et al., 1998). A telomeraseindependent telomere maintenance mechanism, called A L T (alternative lengthening of telomeres), is also evident in certain human cells (usually tumor cells) (reviewed in Henson et al., 2002). 1.3.2 Structure at telomere termini Replication by the conventional replication machinery is predicted to generate structurally asymmetric telomeres, with a blunt end on the leading strand telomere and an overhang on the G-rich strand of the lagging strand telomere (Figure 1-4). The presence of overhangs were first documented in Oxytricha and Euplotes, in which the fixed number of telomeric repeats allowed direct sequencing of both strands (Klobutcher et al., 1981). Using this approach, all telomeres were found to possess a 3' overhang of 16nt or 12nt in Oxytricha or Euplotes respectively (Klobutcher et al., 1981). However, most organisms have a variable number of telomeric repeats, making it impossible to detect overhangs by direct sequencing (Henderson and Blackburn, 1989). A number of different techniques have been employed to characterize telomeric overhangs since then. Using osmium tetraoxide, which preferentially reacts with unpaired thymine residues, Henderson and Blackburn (1989) demonstrated the presence of 3' overhangs of approximately 12-16nt regardless of telomere length in Tetrahymena and Didymium, suggesting that the overhang is a conserved structure of  24  telomeres and may be involved in telomere function (Henderson and Blackburn, 1989). S. cerevisiae was also found to harbour 3' telomeric overhangs by non-denaturing Southern hybridization (Wellinger et al., 1993a; Wellinger et al., 1993b). Furthermore, a circular form of a linear plasmid (CFP) with a single replication origin and telomeric D N A with TG,.  3  overhangs at both ends was detected in late S phase (Wellinger et al., 1993b). The appearance of the circular form of the plasmid is an indication of telomere-telomere associations, likely to be mediated by non-canonical base pairing of the overhangs (Wellinger et al., 1993b). A more detailed study of the formation of CFP by two-dimensional gel electrophoresis confirmed the presence of long 3' overhangs of >50bp on both leading and lagging strand telomeres in a cell cycle-regulated manner (Wellinger et al., 1996). Because a blunt ended telomere is predicted for the leading strand telomere right after replication (Figure 1-4), the presence of overhang on this telomere point to an extra step that removes nucleotides from the 5' strand to generate the overhang (Wellinger et al., 1996). Because the formation of CFP is unaffected in the absence of telomerase, the extra step of telomere processing does not depend on telomerase (Wellinger et al., 1996). Presence of 3' G-rich overhang in multicellular organisms was first demonstrated in human cell lines using different approaches. Makarov et al. (1997) described the presence of 3' overhang in different mortal human cell lines and immortalized fibroblasts by a technique called primer-extension/nick translation (PENT), in which the presence of 3' overhang allows the hybridization of a primer that is extended by Taq polymerase. Because of its polymerization and 5'-to-3' exonuclease activities, Taq polymerase is able to fill the gap between the primer and the 5' end of the C-rich strand, and start to propagate the nick towards the 3' direction, hence the name of the technique (Makarov et al., 1997). Wright et al. (1997) were able to purify non-denatured telomeric restriction fragments by their ability to hybridize to biotinylated oligonucleotides that are complementary to the G-rich telomeric strand, indicating the presence of 3' overhang. In a different experiment, fibroblasts were cultured in bromodeoxyuridine (BrdU) (Wright et al., 1997). Analysis of D N A in which one strand was substituted with BrdU, which is indicative of one round of replication, revealed that the overhang is only present in the lagging strand telomere, contrary to Makarov et al. (1997) who reported that the majority of telomere ends possess an overhang (Wright et al., 1997). Using electron microscopy, Wright et al. (1997) calculated the average length of  25  telomeric overhangs to be ~200bp in human fibroblasts, which is consistent with the estimation of 130-210 bp by Makarov et al. (1997). Non-denaturing Southern hybridization also confirmed the presence of long 3' overhangs in different normal or transformed human cell lines (McElligott and Wellinger, 1997). In addition, it was shown that long overhangs can be observed in non-cycling human cells, unlike yeast, in which overhangs can only be detected in late S phase (McElligott and Wellinger, 1997). The presence of 3' G-rich overhangs in different plant species has also been reported (Riha et al., 2000). The most detailed studies of telomeric 3' overhang to date were carried out in Tetrahymena, in which a ligation-mediated primer extension technique was used to accurately measure the length of the 3' G-rich overhang and to identify the nucleotide at which the overhang terminates (Jacob et al., 2003; Jacob et al., 2001). It was found that the majority of Tetrahymena telomeres terminate in the sequence of 5 T G G G G T 3 ' , and among these telomeres, most carry an overhang of 14-15nt or 20-2 lnt, suggesting that the terminal structure of Tetrahymena telomeres are under tight regulation (Jacob et al., 2001). Unlike yeast, no significant cell cycle-dependent changes in overhang length were observed (Jacob et al., 2001). In a different study, the same technique was used to study the structure of telomere termini in a conditional telomerase knockout strain as well as different telomerase R N A template mutant strains of Tetrahymena (Jacob et al., 2003). In the absence of telomerase, the precision of telomere processing is affected but most telomeres still terminate with 5'TGGGGT3' and an overhang of 14-15nt or 20-2lnt, indicating that telomerase is not essential for correct processing of telomere termini (Jacob et al., 2003). Telomere termini processing in the R N A template mutants is also largely unperturbed (Jacob et al., 2003). Based on all these information, it was proposed that processing of Tetrahymena telomere termini involve both cleavage of the G-rich strand and degradation of the C-rich strand by some telomerase-independent activities that are sequence non-specific (Jacob et al., 2003). 1.3.3 Higher order structure of telomeres The fact that the 3' overhang is a conserved feature of telomere termini, and the involvement of active mechanism(s) in generating the overhangs suggest that they may have functional roles. It is now evident that a 3' overhang is required for formation of a structure, called tloop (Figure 1-5), which is believed to be responsible for the distinction between natural  26  chromosome ends and double-stranded breaks (Griffith et al., 1999). T-loop is a lariat-like structure initially observed by electron microscopy when a model telomere with several kb of human telomeric repeats and a 100 to 200nt 3' overhang was incubated with the telomeric protein TRF2 ( T T A G G G repeat binding factor 2, see below) (Griffith et al., 1999). T-loops were also detected in telomeric DNA-containing restriction fragments that were purified from in v/vo-crosslinked nuclei isolated from primary mouse and human cells and enriched by gel filtration (Griffith et al., 1999). Measurement of the size of t-loops revealed a correlation between t-loop size and telomere length, although a broad size range was observed (Griffith et al., 1999). A model where TRF2, a protein critical for protecting mammalian chromosome ends (see below), mediates the invasion of the overhang into the duplex part of the telomere tract was proposed (Figure 1-5) based on a number of observations: (1) in vitro t-loop formation depends on TRF2 and the single-stranded 3' overhang; (2) the sequence-specific double-stranded telomere-binding protein TRF1 is capable of coating the entire circular segment of the t-loop; and (3) the E. coli SSB (single strand binding protein) can form a protein complex at the loop-tail junction of the t-loop (Griffith et al., 1999). Formation of t-loops could sequester natural chromosome ends, thereby preventing them from being recognized as D N A damage sites (de Lange, 2002). Besides mammals, t-loops have been observed in Oxytricha micronucleus, Trypanosoma brucei, and Pisum sativum (garden pea) (Cesare et al., 2003; Munoz-Jordan et al., 2001; Murti and Prescott, 1999).  t-loop fTTABGQln.  tliiiiiiiiiiiiiiiiiiitiiiilAATCGCjn Figure 1-5. Higher order structure of telomeres. Shown is a vertebrate telomere with T T A G G G telomeric repeats. The terminus ends in a single-stranded 3' G-rich overhang, which loops back and invade the double-stranded region of the telomere. A D-loop is formed where the overhang invades the double-stranded region and resembles a recombination intermediate. (Figure adapted from Wang et a l , 2004) 27  1.3.4 Telomere protection It is now widely accepted that the t-loop structure is fundamental to the protective function of telomeres in many organisms (reviewed in de Lange, 2002). At the human telomere, two proteins have been shown to bind the double-stranded region directly: TRF1  and TRF2  (Broccoli et al., 1997; Chong et al., 1995a). Both of these proteins have a C-terminal DNAbinding Myb motif and a dimerization domain within the N-terminal half, and both are ' expressed ubiquitously (Broccoli et al., 1997; Chong et al., 1995a). However, they differ in that the N terminus of TRF2 is basic while it is acidic in TRF1  (Broccoli et al., 1997; Chong  et al., 1995a). The telomere protection function of TRF2 was revealed by studying a dominant negative allele of TRF2, TRF2  A b A m  , in which the N-terminal basic domain and the  C-terminal Myb motif are deleted (van Steensel et al., 1998). Expression of T R F 2  A b A m  in a  human fibrosarcoma cell line results in failure of endogenous TRF2 to accumulate at telomeres, which can be attributed to formation of a heterodimer ( T R F 2  A b A m  and endogenous  TRF2) that is incapable of binding to telomeres (van Steensel et al., 1998). Expression of TRF2  A b A m  induces growth arrest with phenotypic characteristics of senescence (van Steensel  et al., 1998). Cytogenetic studies led to the conclusion that absence of TRF2 at telomeres leads to end-to-end chromosome fusions, which are indicative of loss of telomere protection (van Steensel et al., 1998). Detailed examinations of chromosome fusions showed that telomeric D N A was preserved at sites of chromosome end fusion, and fusions are most likely to be ligations of ends with telomeric DNA instead of association of 3' overhang by Hoogsteen base-pairing (van Steensel et al., 1998). In addition, inhibition of TRF2 causes loss of the overhang (van Steensel et al., 1998). Hence, it was postulated that TRF2 protects chromosomes by maintaining the correct structure at telomere termini (van Steensel et al., 1998). In an attempt to understand the mechanism of chromosome fusions when TRF2 is inhibited, Smogorzewska et al. (2002) studied mouse embryonic fibroblasts (MEFs) that lack D N A ligase IV, the ligase responsible for non-homologous end-joining (NHEJ), and found that these cells do not accumulate telomere fusions in response to T R F 2  A b A m  . Therefore, it  was concluded that N H E J is the main mechanism by which telomeres fuse after inhibition of TRF2 (Smogorzewska et al., 2002). In addition, inhibition of TRF2 results in a significant reduction of overhang signals even before cells enter S phase, indicating the presence of active mechanism(s) in degrading telomeric overhangs (Smogorzewska et al., 2002). In a  28  normal human fibroblast cell line that expresses TRF2  , most telomere fusions are  chromatid-type fusions, although chromosome-type fusions and sister chromatid fusions were also observed at significant levels (Smogorzewska et al., 2002). The type of fusion observed is related to the cell cycle phase at which it occurs: when fusion takes place before D N A replication, both chromatids will be fused between the duplicated chromosomes (chromosome-type fusion). On the other hand, chromatid-type fusion is indicative of a postreplication event (de Lange, 2002). Therefore, data from Smogorzewska et al. (2002) suggest that telomere fusions can happen in both GI and G2. Bailey et al. (2001) observed something quite different when T R F 2  A b A m  is expressed in human fibrosarcoma cell line. They showed  that all fusions are chromatid-type fusions, suggesting that they only occur after D N A replication. Using chromosome-orientation fluorescence in situ hybridization (CO-FISH), it was demonstrated that fusions are mostly between leading strand telomeres (Bailey et al., 2001). de Lange (2002) suggested that the discrepancy could be due to the difference in checkpoint status between normal and transformed cells. The tumor cell line used in Bailey et al. (2001) is p53-deficient so telomere dysfunction may not induce a prolonged G I arrest. Therefore, any telomere defects in those cells are carried through S phase, and fusions do not occur until after D N A replication, resulting in chromatid-type fusions between deprotected telomeres (Bailey et al., 2001; de Lange, 2002). Collectively, a model of mammalian telomere protection has been proposed (de Lange, 2002): TRF2 is required for maintenance of 3' overhangs and mediates their invasion into the duplex part of telomeres, thus sequestering the natural telomere termini. Inhibition of TRF2 leads to active degradation of the overhangs, leading to immediate deprotection of telomeres due to inability to form tloops. The deprotected telomeres become targets for "repair" by NHEJ, the consequence of which is telomere fusions even though the duplex region of telomeres is largely intact (de Lange, 2002). Tazl (telomere-associated in Schizosaccharomyces pombe) in Schizosaccharomyces pombe is a homolog of human TRF1 and TRF2, and is required for regulating telomere length and protecting chromosome ends (Cooper et al., 1997; Ferreira and Cooper, 2001). Tazl' cells accumulate fusions in a NHEJ-dependent manner when arrested in G I , or when growing logarithmically in the absence of functional homologous recombination (HR) (Ferreira and Cooper, 2001). Protection of S. pombe telomeres also requires Potl (protection  29  of telomeres). Potl was identified from database search for similarity to the a subunit of telomere end binding proteins from Oxytricha and other ciliates (Baumann and Cech, 2001). Potl' suffers immediate chromosomal instability, and telomere deprotection is apparent: telomeric sequences cannot be detected in Southern hybridization, and circularization of all three chromosomes occurs in rare survivors (Baumann and Cech, 2001). A human homolog of Potl was also identified by B L A S T search (Baumann and Cech, 2001). Gel mobility shift assays and competition studies showed that both S. pombe and human Potl proteins bind specifically to the corresponding single-stranded G-rich telomeric strand, suggesting that Potl caps the very end of telomeres (Baumann and Cech, 2001). Detailed studies on the binding specificities of ScPotlp and hPOTl led to the proposal that Potl proteins coat the entire single-stranded region of the telomere (Lei et al., 2002; Lei et al., 2004). Evidence of hPOTl involving in telomere protection has come from R N A i (RNA interference) experiments in human cell lines: inhibition of hPOTl expression results in apoptosis or senescence depending on cell type, with a higher incidence of anaphase bridges and a reduction in 3' overhang signals (Veldman et al., 2004; Yang et al., 2005). Very recently it was demonstrated that overexpression of hPOTl partially rescues the pheno types of TRF2  A b A m  , consistent with the hypothesis that hPOTl is involved in telomere end protection  (Yang et al., 2005). The mechanism of telomere protection seems to be quite different in the budding yeast S. cerevisiae. This may be related to the characteristics of & cerevisiae telomeres: heterogeneous telomeric repeats ( T G ^ ) , relatively short telomeric tracts, and absence of a long 3' overhang in most cell cycle phases other than late S phase (reviewed in Taggart and Zakian, 2003). S. cerevisiae does not have a homolog of Potl. Instead, end protection is provided by the essential protein Cdcl3p (cell division cycle) (reviewed in Taggart and. Zakian, 2003). Cdcl3p was first implicated in telomere function when Garvik et al. (1995) showed by two-dimensional gel electrophoresis that cdcl 3 mutants rapidly accumulate single-stranded D N A at telomere-proximal regions. Cdcl3p binds specifically to singlestranded T G L J D N A (Lin and Zakian, 1996; Nugent et al., 1996). Nugent et al. (1996) identified a second allele of CDC 13 that, unlike the first allele described by Garnik et al. (1995), confers progressive telomere shortening. Furthermore, epitasis analysis suggested that telomere shortening is due to defects in the telomerase pathway (Nugent et al., 1996). It  30  was proposed that the presence of two alleles of CDC13 that confer two distinct phenotypes implies that the gene has two discrete functions, one in protecting chromosome ends, and the other in mediating access of telomerase to telomeres (Nugent et al., 1996). Two additional players have been identified in protecting chromosome ends in S. cerevisiae: Stnlp (suppressor of cdc thirteen) and Tenlp (telomeric pathways in association with stnl) (Grandin et al., 2001; Grandin et al., 1997). Stnl temperature-sensitive mutants accumulate single-stranded D N A in telomere-proximal regions at restrictive temperatures, indicating the role of Stnlp in protecting chromosome ends, although telomere length is also deregulated in the mutant strain (Grandin et al., 1997). Tenlp was identified in a screen for suppressors of temperature-sensitive stnl mutants (Grandin et al., 2001). Stnlp and Tenlp interact in both co-immunoprecipitation and two-hybrid assays (Grandin et al., 2001). A l l three of the characterized mutant Stnlp proteins are defective in interaction with Tenlp, and reestablishment of interaction by fusing the mutant Stnlp and wild-type Tenlp is sufficient to rescue the growth defects and telomere phenotypes in stnl mutant strains, suggesting that the interaction is essential for telomere functions (Grandin et al., 2001). The proposal that Cdcl3p has two separate functions at the telomere was confirmed by a series of experiments where different proteins or protein domains were fused to the high affinity D N A binding domain of Cdcl3p ( D B D  CDC]3  ) (Pennock et al., 2001). Lethality of  cdcl3A can be rescued by expressing a fusion protein between D B D  C D C 1 3  and Stnlp,  indicating that the end protection function of Cdcl3p relies on its ability to recruit Stnlp to telomere ends. However, the telomere shortening phenotype of cdcl3A remains. This phenotype can be alleviated by expressing another fusion protein between D B D  C D C 1 3  and one  component of the telomerase holoenzyme, although telomeres are hyper-elongated in this case, probably due to aberrant regulation of telomerase activity and/or prolonged localization of telomerase at telomere termini. The authors then identified a region in Cdcl3p that is the putative telomerase recruitment domain (RD). B y expressing a fusion between D B D RD, normal telomere length is restored in cdcl3A that also expresses the D B D  CDC13  C D C ] 3  and  -Stnlp  fusion. Therefore, the utilization of different fusion proteins led to the conclusion that wildtype telomeres can be fully reconstituted in a strain that lacks intact Cdcl3p, when two different complexes, one for end protection and the other for telomerase activities, are delivered to telomere ends (Pennock et al., 2001) (Figure 1-6). The effects of Stnlp and  31  Tenlp on telomere length were proposed to be due to "competition for a single recruitment site in Cdcl3p" for Stnlp and telomerase (Lustig, 2001; Pennock et al., 2001).  //  '  Figurel-6. Proposed functions of Cdcl3p in telomere protection and telomere elongation. Cdcl3p has been proposed by Pennock et al. (2001) to recruit two separate complexes to the telomere for end protection and telomere elongation. The complex containing Stnlp is responsible for telomere protection and inhibits degradation of the C-strand of telomeric D N A . The complex containing the various components of the telomerase holoenzyme (Estlp, Est2p, Est3p and the telomerase R N A template are shown in the diagram) is also recruited by Cdcl3p for telomere elongation. (Figure adapted from Pennock et al., 2001)  32  1.3.5 The telomerase ribonucleoprotein complex Eukaryotes have evolved the use of telomeric repeats to prevent replicative loss from affecting coding regions and to distinguish natural chromosome ends from double-stranded breaks. However, attrition of telomeric D N A will eventually shorten telomeres to a length that can no longer support the protection function. The mechanism by which most organisms replenish telomeric D N A is telomerase (reviewed in Blackburn, 2001; Smogorzewska and de Lange, 2004). Telomerase activity was first identified in Tetrahymena cell-free extract (Greider and Blackburn, 1985). Further analyses of the activity showed that the enzyme is a ribonucleoprotein (RNP) complex, and an integral R N A component was hypothesized to be the template for in vivo telomere addition (Greider and Blackburn, 1987). The hypothesis was supported by the identification and cloning of the R N A component (Greider and Blackburn, 1989) and confirmed by mutational studies in which altering the sequence of the R N A component results in changes in the telomeric repeats in vivo (Yu et al., 1990). The size and the primary sequence of telomerase R N A from different organisms varies remarkably; however, the secondary structure of the R N A is well conserved (Collins, 1999; Chen et al., 2000). Secondary structures proposed for telomerase RNAs in divergent groups of ciliates have common characteristics including a single-stranded region containing the template, a pseudoknot, and some stem/stem-loop structures (Collins, 1999; Lingner et al., 1994; Romero and Blackburn, 1991). The overall architecture of the structure proposed for vertebrate telomerase R N A is remarkably similar to the ciliate structure, except the vertebrate telomerase R N A has two extra domains and a more elaborate pseudoknot (Chen et al., 2000) (Figure 1-7). The evolutionary conservation of telomerase R N A structure indicates that it is the structure instead of the primary sequence that defines the biological function of the RNAs (Chen et al., 2000). For example, it has been shown that the pseudoknot is required for the association of R N A with the reverse transcriptase component in both Tetrahymena and yeast (Gilley and Blackburn, 1999; Lin et al., 2004).  33  P6.1  Pseudoknot  Ciliate  d  elix lllb helix I ]=! mill  •3'  fflnrn,"  j Template  template  helix II  Pseudoknot JJ3  Vertebrate  TTIIIIIII  W~ 2  b  —  ~)  fl'TfT  [Template ,'boundary  3-» template  Figure 1-7. Structure of the telomerase R N A is conserved. Conserved features of the telomerase R N A structure include a single-stranded template region, a pseudoknot and a stem-loop. (Figure adapted from Chen and Greider, 2004)  The first cloned telomerase catalytic subunit was reported by Lingner et al. (1997b). One of the two proteins (pl23) purified from active telomerase complex from Euplotes aediculatas was digested with trypsin, and the resulting peptides were sequenced by mass spectrometry (Lingner et al., 1997b). From the peptide sequences, the authors were able to identify an open reading frame (ORF) encoding a 1031 amino acid protein. B L A S T search showed that one of the closest matches was S. cerevisiae Est2p (ever shorter telomeres), which was shown previously to be required for telomere maintenance (Lendvay et al., 1996) Examination of the sequence of the two proteins revealed motifs in the central domain that are conserved in all reverse transcriptases (RTs) (Xiong and Eickbush, 1990). Substitutions in the most conserved residues in the RT domain of Est2p result in short telomeres and senescence in yeast, and also abolish in vitro telomerase activity, indicating telomerase is a  34  RT (Lingner et al., 1997b). Independently, Counter et al. (1997) also identified Est2p as the catalytic subunit of telomerase in S. cerevisiae. Subsequently, telomerase reverse transcriptase (TERT) was identified in S. pombe and human (Nakamura et al., 1997). Sequence alignment of the four TERTs (Euplotes pl23, S. cerevisiae Est2p, S. pombe Trtlp, and human hTERT) revealed they all have similar modular organization, with a central R T domain containing all 7 RT motifs (1, 2, A , B ' , C, D, E), a long N-terminal domain with conserved motifs, and a short, less conserved C-terminal domain (Nakamura et al., 1997) (Figure 1-8). Mutational analyses have confirmed that the most conserved residues in the R T domain are critical for telomerase activity in different organisms (Counter et al., 1997; Haering et al., 2000; Lingner et al., 1997b; Weinrich et al., 1997). Furthermore, based on previous studies on processivity of HIV-1 (human immunodeficiency virus type 1) RT, it was found that the corresponding residues in the C and E motifs of the RT domain in Est2p also influence processivity, which in turn affects telomere length (Bryan et al., 2000a; Peng et al., 2001). Outside of the RT domain, several motifs have been identified in the N-terminal that are also conserved among almost all characterized TERTs. B y mutagenizing a plasmid-borne EST2 gene and carrying out complementation in an est2 mutant strain, Friedman and Cech (1999) identified four essential domains in the N-terminal (I to IV) by analyzing the resulting artificial phylogeny of mutant alleles. Similarly, Xia et al. (2000) also identified four conserved domains from alignment of all known TERTs. These four domains, GQ, CP, QFP, and T (Figure 1-8), overlap with domains I to IV as defined by (Friedman and Cech, 1999), suggesting a high level of conservation in the N-terminus of TERTs (Xia et al., 2000). Mutational analyses have identified the N-terminal to be crucial for binding telomerase R N A in yeast, Tetrahymena, and human (Armbruster et al., 2001; Bachand and Autexier, 2001; Bryan et a l , 2000b; Friedman and Cech, 1999; Lai et al., 2001). Friedman and Cech (1999) and X i a et al. (2000) noticed a number of alanine substitution mutations in the N terminal domain of Est2p that are unable to support in vivo telomere maintenance, but none the less display in vitro telomerase activity. Armbruster et al. (2001) investigated this further by generating a panel of blocks of substitution mutations in the N-terminal domain of hTERT and identified a region called D A T (dissociates activities of telomerase) within motif I of the N-terminal domain that is dispensable for in vitro enzyme catalysis but essential for biological activity. The function of D A T is unclear but it has been implicated in association  35  between telomeres and telomerase (Armbruster et al., 2003). The C-terminal domain is less conserved, but deletion analyses showed that the domain is also required for telomerase activity (Bachand and Autexier, 2001; Lai et al., 2001) and processivity (Peng et al., 2001).  M. musculus  *thaHina  - —  1  ' i i ^ — •  I  If  I  (.  j-f  f-  •  C elegans Figure 1-8. Modular structure of telomerase reverse transcriptase (TERT). The protein can be divided into three domains: the N-terminal domain, the central reverse transcriptase (RT) domain, and the C-terminal domain. The RT domain consists of the seven motifs conserved among all reverse transcriptase. The relatively large gap between motifs A and B ' is a unique feature of TERT. The N-terminal domain carries several motifs (GQ, CP, QFP, T) that are conserved in most TERTs. The C-terminal domain is the least conserved. The C. elegans TERT has a relatively small N-terminal domain, and lacks the C-terminal domain (Malik et al., 2000). Malik et al. (2000) noted that motif T and a fragment that overlaps with motif GQ are missing in the C-terminal domain of C. elegans TERT. There has been no documentation of other conserved motifs in this particular TERT. (Figure modified from X i a et al., 2000)  36  It is believed that TERTs belong to a distinct group of RTs based on a number of features of TERTs: (1) there are conserved motifs outside of the RT domains that are exclusive to TERTs; (2) a large gap is present between RT motifs A and B ' in all TERTs; (3) within the TERT RT domain, there are several substitutions of amino acids that are highly conserved in other RTs (Eickbush, 1997; Nakamura et al., 1997). Variations within the RT domain between TERTs and other RTs could reflect the mechanistic difference between the two: RTs are capable of reverse transcribing a long stretch of R N A , while TERTs only copy a small, well defined region of its R N A template (Nugent and Lundblad, 1998). Most TERTs have the organization depicted in Figure 1-8. However, two exceptions have been noted (Malik et al., 2000). TERT from Giardia lamblia lacks some of the conserved motifs in the N-terminal domain, even though its size is comparable to other TERTs. TERT from C. elegans is surprisingly small, being only two-thirds to one-half of the size of other TERTs. Furthermore, the C-terminal domain is absent; the N-terminal domain is truncated, and no conserved motifs have been found so far (Figure 1-8) (Malik et al, 2000). In both G. lamblia and C. elegans TERTs, several highly conserved residues in the RT motifs are substituted, which is expected to affect the enzymatic properties of the corresponding TERT significantly (Malik et al., 2000). Phylogenetic analysis suggests that G. lamblia TERT is the most diverged, and C. elegans TERT may be the closest to the ancestor of the rest of the TERTs (Malik et al., 2000). 1.3.6 Consequences of telomerase deficiency A number of model organisms have been employed to study the biological consequences of telomerase depletion, by knocking out the expression of the reverse transcriptase component, the telomerase R N A , or other components that are required for in vivo telomerase activity. In S. cerevisiae, besides the genes encoding the telomerase reverse transcriptase (JEST2) and the telomerase R N A (TLC1, for telomerase component 1), mutations in three additional genes (EST1, EST3, CDC13) also lead to progressive telomere shortening at a rate of ~34nt/generation and senescence, which are phenotypes expected for telomerase deficiency (Counter et al., 1997; Lendvay et al., 1996; Lingner et al., 1997a; Lundblad and Szostak, 1989; Singer and Gottschling, 1994). Hackett et al. (2001) carried out a detailed examination of genomic instability in estlA, whose deficiency in in vivo telomerase activity is believed to  37  be a result of failure to recruit telomerase to telomeres or to activate the enzyme in vivo (Evans and Lundblad, 1999; Taggart et al., 2002). Mutation rates were measured and the types of mutations were monitored by using reporter genes that allowed selection or colour screening (Hackett et al., 2001). It was found in estlA, mutation rate is comparable to wildtype in early generations, but increases ten-fold in late generations when most cells die off (Hackett et al., 2001). Frequent chromosomal rearrangements involving terminal but not internal sequences were detected (Hackett et al., 2001). Furthermore, sequencing revealed no telomeric sequences were present at the fusion junctions (Hackett et al., 2001). These observations reaffirm the notion that telomerase promotes genomic stability (see below). In Arabidopsis thaliana, a telomerase deficient strain (AtTERT-/-) loses telomeric D N A at a rate of 250-500bp per generation, and mutants are able to survive for up to 10 generations (Fitzgerald et al., 1999; Riha et al., 2001). In all generations analyzed, T R F analysis gave rise to discrete bands that correspond to individual chromosome ends, instead of a smear of 2 to 4kb as observed in wild-type (Fitzgerald et al., 1999; Riha et al., 2001). Visually, AtTERT-/- is indistinguishable from wild-type up to the fifth generation (G5), after which developmental abnormalities, such as undeveloped anthers with few pollen grains, become apparent and worsening gradually in successive generations (Riha et al., 2001). The appearance of these abnormalities coincides with the emergence of chromosomal instability. While no anaphase bridges were observed before G5 in AtTERT-/-, low levels were observed in G6, and the frequency increased in successive generations (Riha et al., 2001). A more detailed cytogenetic study carried out in G8 AtTERT-/- showed that the mutants have a remarkably high degree of genomic instability, with 50% of nuclei containing anaphase bridges; moreover, complex genome rearrangements suggestive of multiple breakage-fusionbridge cycles were evident (Siroky et al., 2003). Heacock et al. (2004) examined the relationship between telomere length, loss of telomere functions, and formation of telomere fusions in AtTERT-/- with a molecular approach. A PCR-based technique called primer extension telomere repeat amplification (PETRA) was devised for measuring telomere length on individual chromosome ends (Heacock et al., 2004). Briefly, an adaptor oligonucleotide with a tag on its 3' terminus is hybridized to the 3' G-rich overhang, and primer extension is carried out with D N A Poll (which has 5' —> 3' exonuclease activity). Following primer extension, a specific chromosome end is amplified by PCR using a unique subtelomeric  38  primer and a primer whose sequence is identical to the tag on the adaptor. PCR products are detected by Southern hybridization. Consistent with TRF analysis, P E T R A detected sharper bands in AtTERT-/- relative to wild-type, indicating that the size distribution of telomeric D N A in an individual plant is dramatically reduced when telomerase is depleted (Heacock et al., 2004). To detect potential telomere fusions, PCR was carried out using different combinations of subtelomeric primers (Heacock et al., 2004). It was found that more PCR products were amplified in G6, G7, and G9 compared to G5 or wild-type. Therefore, the presence of telomere fusions and the onset of cytogenetic abnormalities seemed to occur concurrently (Riha et al., 2001; Heacock et al., 2004). Cloning and sequencing of fusions revealed that the majority of fusions involve a telomeric end and a subtelomeric end, suggesting that dysfunctional telomeres undergo exonuclease processing before end-joining (Heacock et al., 2004). The mouse model for the consequences of telomerase deficiency has been studied extensively. A mouse strain in which the entire telomerase R N A gene (mTR) is deleted remains viable for six generations (Blasco et al., 1997). Quantitative fluorescence in situ hybridization (Q-FISH) on primary mouse embryonic fibroblasts (MEFs) derived from different generations of mTR-/- shows that telomeres shorten at a rate of 4.8 ± 2.4kb per generation (Blasco et al., 1997). As generation number progresses, the percentage of cells with aneuploidy, telomeres without detectable repeats, or end-to-end associations/fusions increases (Blasco et al., 1997; Hande et al., 1999; Hao and Greider, 2004). Closer examination of MEFs from G6 mTR-/- by Q-FISH and chromosome painting revealed that most end-to-end fusions involve two p arms with no detectable telomeric sequences at fusion junctions, again providing support for the association between telomere shortening and chromosomal instability (Hande et al., 1999). Phenotypic analyzes of mTR-/- mice revealed no adverse effect in overall fitness in first-generation (GI) mice, but significant decline in litter size becomes apparent in G4 (Lee et al., 1998). Progressive germ cell depletion by both decreased proliferation and increased apoptosis results in small testes in G6 animals (Lee et al., 1998). Pathological changes associated with aging were also observed in late generation mTR-/- mice at a young age: hair graying and loss, skin lesions, reduced capacity to cope with acute stress such as wound healing, and notably a four- to six-fold increase in the incidence of spontaneous cancers compared to age-matched mTR+/+ mice (Rudolph et al.,  39  1999). Cytogenetic analysis showed a significant increase in the incidence of chromosome fusions and aneuploidy in tumors derived from mTR-/- mice relative to those derived from mTR+/+ mice (Rudolph et al., 1999). These observations are consistent with the notion that genomic instability resulted from loss of telomere function facilitates tumorigenesis (Rudolph et al., 1999). Chromosome fusions from G5 and G6 mTR-/- were also characterized molecularly, and it was found that in all cases no telomeric repeats were present at the fusion junction (Hemann et al., 2001). A mouse strain with disrupted TERT (mTert-/-) and no detectable telomerase activity was generated subsequently (Liu et al., 2000). Splenocytes from progressive generations and embryonic stem (ES) cells display the expected progressive telomere shortening phenotype upon culturing (Erdmann et al., 2004; Liu et al., 2002). Chromosomal fusions and increased frequency of aneuploidy are observed only in later passages of mTert-/- ES cells (Liu et al., 2000). Therefore, the temporal lag between telomere loss and genomic instability is characteristic of telomerase null mutants in yeast, plant, and mouse. Studying telomerase-null mice has revealed that telomerase preferentially elongates short telomeres. Hemann et al. (2001) crossed mTR-/- G6, which had short telomeres, to mTR +/-, which had long telomeres. Such intergenerational cross produced mTR+/-(iFl) and mTR-/-(iFl), both of which inherited short telomeres from the mTR-/- G6 parent and have similar average telomere length. However, only mTR-/-(iFl) displayed reduced testes weight, testicular apoptosis, and increased frequency of chromosome fusions as in mTR-/- G6. Importantly, mTR-/-(iFl) but not mTR+/-(iFl) had a high frequency of very short telomeres or signal-free ends, suggesting that telomere function in mTR+/-(iFJ) was restored by addition of telomeric repeats to the shortest telomeres by telomerase. The preference to elongate short telomeres is also evident upon prolonged culturing of mTert +/- ES cells (Liu et al., 2002). These cells progressively lose telomeric D N A , indicating haploinsufficiency in mTert. However, unlike mTert -/- cells, mTert+/- cells do not exhibit an increased frequency of critically short telomeres or genomic instability (Liu et al., 2002). A higher probability of short telomeres being elongated by telomerase has been demonstrated recently in S. cerevisiae, whose heterogeneous telomeric repeats allow development of an experimental system that can distinguish between telomeric D N A synthesized by semi-conservative D N A replication and telomeric D N A elongated by  40  telomerase at individual telomere ends (Teixeira et al., 2004). A number of characteristics of telomere elongation by telomerase were revealed: (1) Not every telomere is extended in each cell cycle; (2) the length of telomeric D N A being added in each cell cycle is very heterogeneous, ranging from a few to a few hundred nucleotides; (3) the length of added telomeric D N A does not correlate with the original telomere size; (4) the frequency of telomere elongation correlates with the original telomere size (Teixeira et al., 2004). These results imply that a typical telomere escapes elongation by telomerase for several cell cycles. It was therefore proposed that "telomeres switch between telomerase-extendible and telomerase-nonextendible states" (Teixeira et al., 2004). It becomes apparent that besides maintaining average telomere length, telomerase may promote the protective function of telomeres. Indirect evidence includes the fact that some normal human cells possess measurable telomerase activities, yet their telomere lengths continue to shorten with propagation in culture (Broccoli et al., 1995; Liu et al., 1999). In addition, chromatin immunoprecipitation revealed that yeast Est2p (reverse transcriptase component) is associated with telomeres even when they are not being elongated, leading to the proposal that Est2p may protect chromosome ends when it is not actively elongating telomeres (Taggart et al., 2002). A more direct demonstration that telomerase offers protection other than telomere length maintenance has come from studying normal human fibroblasts, which have no detectable in vitro telomerase activity (Harley, 1991; Masutomi et al., 2003). However, using a monoclonal antibody that recognizes catalytically active human TERT (hTERT), Masutomi et al. (2003) were able to observe low levels of active telomerase in early passage human fibroblasts during their progression through S phase. Inhibition of telomerase in these cells leads to slow proliferation and early entrance into senescence, despite the fact that there is no significant difference in telomere length between telomeraseinhibited and control fibroblasts (Masutomi et al., 2003). Furthermore, telomerase inhibition also reduces overhang signals in the cells, as detected by the assay developed by Stewart et al (2003) (Masutomi et al., 2003). Therefore, periodic expression of telomerase may promote cellular survival and telomere integrity in normal human cells (Masutomi et al., 2003). These findings are related to several previous studies which suggested that altered telomere structure, instead of short telomere length, is the trigger of replicative senescence: first, cells that overexpress TFR2 senesce at much shorter telomere length (Karlseder et al., 2002);  41  second, loss of 3' overhang is correlated to replicative senescence (Li et al., 2003; Stewart et al., 2003). 1.3.7 Telomere length regulation Even though altered telomere structure is likely to be the trigger of replicative senescence and D N A damage response, it is important to consider telomere length when assessing telomere functioning, because a minimum length is required for maintaining the structure (e.g. t-loop), and alteration of telomere length homeostasis could be an indication of deregulation in telomere metabolism (reviewed in Blackburn, 2001). Telomere length is normally kept within a well-defined range that is organism- and strain-specific (Kipling and Cooke, 1990; Shampay and Blackburn, 1988). How the cell maintains average telomere length within certain limits has been studied mainly in yeast and human cell cultures. In yeast, the major protein that is involved in telomere length regulation is Raplp (repressor/activator protein 1). Raplp is an essential, multifunctional protein that binds specific D N A sequences found in a large number of genetic loci, although C1.3A is among one of the highest affinity sites (reviewed in Shore, 1994). Footprinting experiments suggest that Raplp could bind telomeric D N A up to once every 18bp, meaning -20 molecules could potentially bind a telomeric tract with a typical length (Gilson et al., 1993). A functional role of Raplp at telomeres was first revealed by telomere length changes when RAP1 is mutated or overexpressed in S. cerevisiae (Conrad et al., 1990; Kyrion et al., 1992; Lustig et al., 1990). The C-terminal domain of Raplp is crucial for the telomere length regulation function of the protein, as a truncation mutant that lacks this domain has vastly increased telomere length, and tethering of the C-terminal domain of Raplp to sites adjacent to telomeric tract also affects telomere length (Kyrion et al., 1992; Marcand et al., 1997). To investigate the mechanism of Raplp regulating telomere length, Marcand et al. (1997) devised a system where different numbers of Gal4p-binding sites were inserted adjacent to a telomeric tract so that there is a small linker between these sites and the beginning of the telomeric tract. These strains were also engineered to express a hybrid protein in which the C-terminal domain of Raplp is fused to the DNA-binding domain of Gal4p. Therefore, the C-terminal domain is tethered to an internal site very close to the telomere (~40bp away). It was found that the strains have reduced telomere length. Significantly, the more Gal4-  42  binding sites are inserted, the larger the extent of decrease in telomere length. Based on these results and the fact that multiple Raplp molecules could bind a telomeric tract, the authors proposed a negative feedback model, in which the number of Raplp molecules bound to the end of a chromosome is counted and actively maintained to regulate telomere length (Marcand et al., 1997). Two Raplp-interacting factors, Riflp and Rif2p, affect telomere length in a similar manner as the C-terminal domain of Raplp and interact with one another (Hardy et al., 1992; Wotton and Shore, 1997). Recently, using a similar system as Marcand et al (1997), it was shown that Riflp and Rif2p can be counted (i.e. displaying decreasing telomere length with increasing Gal4p-binding sites inserted adjacent to the telomere) in the absence of the C-terminal domain of Raplp, and that counting of Raplp is dependent on the R i f proteins (Levy and Blackburn, 2004). Therefore, it appears that it is the number of the R i f proteins that is sensed, allowing negative regulation of telomere length (Levy and Blackburn, 2004). It appears that humans also employ a similar counting mechanism in maintaining telomere length within set limits as in yeast. The double-stranded telomeric D N A binding protein TRF1, is a negative regulator of telomere length in human cell culture (Chong et al., 1995a; Smogorzewska et al., 2000; van Steensel and de Lange, 1997). Therefore, TRF1 is functionally similar to yeast Raplp. However, a large number of recent studies suggest that the situation in humans may be a lot more complicated and involves interplay among many telomeric proteins (Colgin and Reddel, 2004; Smogorzewska and De Lange, 2004) (Figure 19). TIN2 (TRF1-interacting nuclear protein 2) was first identified in a yeast two-hybrid screen using TRF1 as a bait (Kim et al., 1999). A truncation mutant of TIN2 confers telomere elongation, indicating a role in telomere length regulation (Kim et al., 1999). TIN2 has been implicated in multiple aspects of telomere length regulation. First, it has been shown that TIN2 controls inhibition of TRF1 by the protein tankyrase 1, which has poly(ADP-ribose) polymerase (PARP) activity and removes TRF1 from telomeres in vivo by the formation of poly(ADP-ribose) (Smith and de Lange, 2000; Smith et al., 1998; Y e and de Lange, 2004). Second, TIN2 interacts with PTOP/PIP1/TINT1 (POT1- and TIN2-organizing protein / POT 1-interacting protein 1 / TIN2-interacting protein 1), which is believed to recruit POT1 to telomeres (Houghtaling et al., 2004; Liu et al., 2004b; Y e et al., 2004b). POT1 was first identified in S. pombe as a single-stranded telomeric D N A binding protein and is required for  43  telomere protection (Baumann and Cech, 2001). Human POT1 was later shown to associate with TRF1 and influence telomere length in cell cultures, leading to the speculation that POT1 transmits information about telomere length (through its interaction with TRF1) to telomerase, which acts on the single-stranded telomere terminus (Loayza and De Lange, 2003). Third, TIN2 binds both of the double-stranded telomeric DNA-binding proteins, TRF1 and TRF2, and appears to be important for the stabilization and localization of both proteins (Houghtaling et al., 2004; K i m et al., 2004; Liu et al., 2004a). Gel filtration analysis showed co-elution of all the above proteins, suggesting that telomeric proteins exist in a multi-subunit complex (Liu et al., 2004a; Y e et al., 2004a).  Stable T-loop  Figure 1-9. Telomere length is regulated by multiple proteins in humans. Results from a number of studies suggest that information about the length of a telomere may be transmitted to the telomere terminus by a complex of proteins involving TRF1, TIN2, PTOP/PIP1/TINT1, and POT1 (reviewed in Colgin and Reddel, 2004). (Figure adapted from Colgin and Reddel, 2004)  1.4 G-rich D N A in C. elegans 1.4.1 C. elegans as a model organism Several characteristics of C. elegans make it an excellent model organism (Brenner, 1974; Jorgensen and Mango, 2002): (1) it is small and has a rapid life cycle (~3 days at 20°C), so a large number of animals can be handled and examined for genetic studies; (2) the  44  hermaphrodites reproduce mainly by self-fertilization but are also capable of crossing to males, allowing simple strain construction strategies; (3) progression of meiosis within the gonad can be observed readily; (4) C. elegans has an invariant somatic cell lineage, with each hermaphrodite having exactly 959 somatic cells (Sulston and Horvitz, 1977); (5) the genome size is relatively small (~ 100Mb), facilitating the complete sequencing of the entire genome (C. elegans Sequencing Consortium, 1998). Genetic screens to identify mutations that give rise to specific phenotypes are well established (reviewed in Jorgensen and Mango, 2002). Development of the gene expression inhibition technique, R N A interference (RNAi) (Fire et al., 1998), has also helped in identifying genes linked to human cancers (Poulin et al., 2004). Because of the short life cycle and small body size, C. elegans can be used for measuring spontaneous mutation frequencies. Denver et al. (2004b) have provided a direct estimation of spontaneous mutation rate by direct sequencing to detect mutations that have accumulated over >300 generations. A rate of ~2.1 mutations per genome per generation was calculated, which was an order of magnitude higher than previous indirect estimates based on mutations that affect phenotypic traits (Denver et al., 2004b; Drake et al., 1998; reviewed in Keightley and Charlesworth, 2005). These estimates apply only to non-lethal mutations. The frequency of spontaneous recessive lethal mutations has also been measured using a genetic system in which de novo lethal mutations within crossover-suppressed region(s) can be maintained in a heterozygous state (Rosenbluth and Baillie, 1981, Rosenbluth et al., 1983). Because the lethal mutations are pseudo-linked to genetic markers within the crossoversuppressed region, they can be detected by absence of the genetic markers in the progeny. Therefore, by setting up a large number of worms of the test strain and screening for absence of the genetic markers in the progeny, one can estimate the frequency of lethal mutations within the crossover-suppressed regions (Rosenbluth et al., 1983). In wild-type background, it has been estimated that the frequency is 0.06% within the left half of chromosome V and the right half of chromosome III (regions in which crossover is suppressed by the reciprocal translocation eTl) (Rosenbluth et al., 1983). 1.4.2 Features of (G/C)n in the C. elegans genome The C. elegans genome, like other eukaryotic genomes, is enriched in mononucleotides (see section 1.1.1). Denver et al. (2004a) carried out a detailed survey of mononucleotides longer  45  than or equal to 8bp in the C. elegans genome and revealed features that have not been described previously in other genomes. Although the number of (A/T)n vastly exceeds that of (G/C)n, the degree of over-representation of (G/C)n is in fact greater than that of (A/T)n, based on the A+T to G+C ratio of 2:1 in C. elegans (Denver et al., 2004a). The distribution of mononucleotides along the chromosome is not even in all of the autosomes. Mononucleotides are denser in the autosomal arm regions than in the central core regions (Denver et al., 2004a). This uneven distribution was observed for all four types of mononucleotides (A, T, C or G on the + strand) (Denver et al., 2004a). Clusters of mononucleotides are apparent (defined as >300 tracts per lOOkb), most of which are in the autosomal arms, while core regions display a baseline density of-90 to 100 tracts per lOOkb (Denver et al., 2004a). B y contrast, chromosome X has a more uniform distribution of mononucleotides and is largely devoid of high-density clusters (Denver et al., 2004a). Autosomal arm regions in the C. elegans genome are in general rich in simple repeat sequences and low in gene density (C. elegans Sequencing Consortium, 1998). In addition, they have been shown to have increased frequency of recombination, suggesting that recombination may contribute to the observed distribution bias (Barnes et al., 1995; Brenner, 1974). Denver et al. (2004) also observed a spacing pattern of mononucleotides that is strongly biased towards closely spaced (0 to 50 intervening nucleotides) tracts. In other words, two adjacent mononucleotide tracts separated by 0 to 50 nucleotides are more frequent than those separated by more than 50 nucleotides. Furthermore, among mononucleotide pairs that have no intervening nucleotides, (A)n(T)n pairs immensely outnumber (T)n(A)n pairs (on the + strand) for some unknown reasons (Denver et al., 2004a). A striking feature is the difference in abundance of tracts of different lengths between (A/T)n and (G/C)n: (A/T)n become over-represented above a tract length of 3bp, but abundance continues to decline with increasing tract length; by contrast, the observed abundance of (G/C)n matches the expected abundance up to a tract length of 8bp, above which their abundance remains relatively constant and actually becomes more abundant than (A/T)n beyond a tract length of 15 (Denver et al., 2004a). The abundance of long (G/C)n (>17nt) seems to be a feature of the C. elegans genome that is not shared by many other genomes.  46  1.4.3 Microsatellite instability in C. elegans mutants Maintenance of mononucleotides and other types of microsatellites in C. elegans has been shown to depend on the mismatch repair pathway as in other organisms. Degtyareva et al. (2002) showed that disruption of msh-2 (a MutS homolog) by insertion of a transposable element (Tel) leads to increased spontaneous mutation rates, reduced survival, reduced apoptosis induced by D N A damage in germ cells, and instability of different microsatellites. Using PCR and sequencing gels, the authors were able to detect significantly increased frequencies of alterations in tract lengths of (GT)n, (AAT)28, and (AAAT) 3 compared to 4  wild-type (Degtyareva et al., 2002). Consistent with previous studies on the relation between tract length and stability, frequencies of changes in (GT)n increase with tract length (Degtyareva et al., 2002). Another MutS homolog, msh-6, has also been shown to affect microsatellite stability in C. elegans (Tijsterman et al., 2002). Similar to msh-2, msh-6 has increased spontaneous mutation rates and instability of microsatellites, including (A) 15, (GT)ig, (GAG)is, and (CTT)i3. Tijsterman et al. (2002) devised a system for visualizing somatic repeat instability. Changes in tract length of (A)n result in expression of lacZ and can be detected by staining with X-gal. It was observed that in msh-6 mutants, msh-2 R N A i , and msh-6 R N A i , a much high percentage of worms had patches of X-gal staining than in wild-type (Tijsterman et al., 2002). Both Degtyareva et al. (2002) and Tijsterman et al. (2002) did not detect any elevated X-chromosome non-disjunction in the mismatch repair mutants, indicating that the mutants do not suffer from chromosomal instability. Another MutS homolog in C. elegans, HIM-14 (high incidence of males), has been shown to promote formation of meiotic crossover but has no apparent role in M M R : mutants display no increased germline spontaneous mutation frequency or microsatellite instability (Zalevsky et al., 1999). 1.4.4 Telomere defects in C. elegans mutants Chromosomal instability could be a consequence of defects in telomere maintenance, as discussed in section 1.3. The first characterization of telomeres in C. elegans was carried out by Wicky et al. (1996), who showed that they are composed of T T A G G C repeats that span between 4 and 9 kb. Using probes derived from the subtelomeric region of different chromosome ends, it was shown that certain ends have unique sequences in their  47  subtelomeric regions (Wicky et al., 1996). For other chromosome ends, the subtelomeric probe is able to hybridize to D N A fragments that are insensitive to Bal3\ digestion, suggesting that they can be found in internal genomic sites (Wicky et al., 1996). Southern hybridization with different subtelomeric probes also revealed chromosome-specific telomere lengths (Wicky et al., 1996). The first C. elegans gene that was demonstrated to be required for telomere maintenance is mrt-2 (mortal germline), which is a highly conserved D N A damage checkpoint gene (Ahmed and Hodgkin, 2000). Mrt-2 mutants display progressive telomere shortening and late onset end-to-end chromosome fusions, as evident by the decreased number of chromosomes detected by DAPI (diamidinophenolindole) staining and pseudo-linkage between genetic markers located on different chromosomes in late generations (Ahmed and Hodgkin, 2000). Mrt-2 is also hypersensitive to X-rays but not U V light (Ahmed and Hodgkin, 2000). Telomere length regulation in C. elegans is not well understood, and only two genes have been implicated in this process. Purified HPR-1 (hnRNP A l homolog) binds C. elegans single-stranded G-rich telomeric repeats in vitro (Joeng et al., 2004). Overexpression of hpr1 leads to elongated telomeres, and this elongation can be reversed upon elimination of hpr-1 overexpression (Joeng et al., 2004). Therefore, it was concluded that hpr-1 participates in telomere length regulation in C. elegans (Joeng et al., 2004). Another C. elegans gene that has been implicated in telomere homeostasis is clk-2 (clock), although its effects on telomere length have been controversial (Ahmed et al., 2001; Benard et al., 2001; L i m et al., 2001). Benard et al. (2001) reported that clk-2 has long telomeres relative to wild-type as analyzed by Southern hybridization to either a telomeric probe or different subtelomeric probes. Furthermore, expression of wild-type C L K - 2 in the clk-2 background results in shortened telomeres (Benard et al., 2001). Concurrently, Lim et al. (2001) described the presence of long telomeres in wild-type that were detected as hybridization to large terminal restriction fragments. These large fragments are absent in the same clk-2 strain, and average telomere length is also shorter in the mutants. Hence, the authors concluded that clk-2 has shorter telomere lengths (Lim et al., 2001). Ahmed et al. (2001) crossed clk-2 to wild-type, and from the heterozygous F I , they set up multiple lines of homozygous and wild-type F2 progeny. In contrary to Benard et al. (2001) and Lim et al. (2001), Ahmed et al. (2001) did not observe any significant difference in telomere length between clk-2 and wild-type. Moreover, no  48  consistent change in telomere length was observed over the generations. According to Ahmed et al. (2001), the difference in telomere length between wildtype and mutant reported by Benard et al. (2001) and Lim et al. (2001) could be attributed to fluctuations in telomere length inherent to C. elegans. 1.5 Thesis objectives Telomere length differs dramatically between two inter-fertile mouse species, Mus musculus (>25 kb) and Mus spretus (5-15 kb) (Kipling and Cooke, 1990; Starling et al., 1990; Zhu et al., 1998). This difference has been shown to be genetically determined (Zhu et al., 1998): F l progeny derived from crossing between a M. musculus parent and a M. spretus parent had intermediate average telomere length. When F l was backcrossed to the M. spretus parental strain, segregation of phenotype was observed, where it appeared that telomeres derived from the M , spretus parent were elongated in some F2's, but remained largely unchanged in the others (Zhu et al., 1998). Mapping data suggested that the genetic factor(s) is/are located within a 5cM region on distal chromosome 2. In an attempt to identify the gene(s) that is/are responsible for the shortened telomeres in M. spretus, Mike Schertzer noticed that one of the candidate genes was very similar to F33H2.1 in the C. elegans genome. At the time a knockout allele of F33H2.1 (gklO) had been generated (the C. elegans Gene Knockout Laboratory at the University of British Columbia, Vancouver, Canada), but no immediate phenotype was observed (Vijayaratnam, 2000). As it turned out, spontaneous mutations occurred at a high frequency in the C. elegans knockout strain, indicative of genomic instability. Therefore, the objectives of my thesis were to investigate the basis of genomic instability in the strain, and to determine i f there is an accompanying telomere phenotype. In the following chapters, I will describe the characterization of genomic instability in the knockout strain, a model of how genomic instability arises in it, the development of a new technique for examining telomere length, and utilization of the technique in examining telomere length in the gklO strain and other mutants.  49  CHAPTER 2  dog-1 is required for the maintenance of G-rich D N A in C. elegans  2.1 Introduction The allele gklO carries a deletion within the clone F33H2.1, which contains a gene encoding a putative helicase known as DOG-1. Dog-1 (gklO) has no visible phenotype. However, upon careful examination over a number of generations, a mutator phenotype could be observed. In this chapter, the mutator phenotype of dog-1 (gklO) will be described. The molecular basis of the mutator phenotype in dog-1 (gklO) was examined and was found to be related to repeat instability. A model of how mutation in dog-1 could lead to the mutator phenotype will be presented.  2.2 Materials and Methods 2.2.1 Strains Worms were handled as described (Brenner, 1974) but were grown at room temperature (19°C to 23°C) unless stated otherwise. The transgenic strain KR3847 was constructed by Ann M . Rose using dog-l(gklO) males (KR3792) and a dpy-5 marked dog-1 (gklO) strain (KR3838). Outcrossed males were crossed to a transgenic strain carrying cosmid ZK340, which contains F33H2.1, as a transgenic array (BC5865). Resulting Roller progeny, which is an indication of inheritance of the transgenic array, were subsequently progeny tested for homozygosity of gklO using PCR as described in section 2.2.3. KR4037 (dog-l(gklO) glp-4(bn2ts)) was constructed from KR1309 (glp-4(bn2ts) unc54(e657)) and KR3613 (dog-1 (gklO)). Heterozygous dog-1 (gklO) males were crossed to glp4(bn2ts) unc-54(e657). Over 100 wild-type-looking F2's were set up from F l ' s that were heterozygous for gklO. These wild-type-looking F2's were screened for homozygous glp4(bn2ts) by shifting 10 wild-type-looking F3 progeny to 25°C and checking for sterility in all  A version of this chapter has been published. Cheung, I., Schertzer, M . , Rose, A . , and Lansdorp, P. M . (2002). Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich D N A . Nat Genet 31,405-409. 1  50  progeny. The genotype of such recombinants should be glp-4(bn2ts) + dog-1 (gklO)Zglp4(bn2ts) unc-54(e657) +. dog-l(gklO) glp-4(bn2ts) was generated by self-crossing of the recombinants.  2.2.2 Worm lysis Single worms were picked into 5uL of lysis buffer (50mM Tris pH8.3, 35uM N a H P 0 , 2  4  0.05% Tween-20, 0.75 m M M g C l , 0.06 mg/ml Proteinase K) in PCR tubes. Worms were 2  frozen at -70°C for 15 minutes or more and then lyzed at 57°C for 60 minutes followed by incubation at 95°C for 15 minutes to inactive the enzyme. 2.2.3 Genotyping of dog-1 strains To check the genotype of dog-1 strains, three primers were used: IL, IR, and IC2. The sequences of these primers were: IL, 5 ' - T G T C C A T T G G G C A C A G A G T A - 3 ' ; IR, 5'G T T G T G A A A G A G G A G C A G C C - 3 ' ; IC2, 5 ' - T C T C C T G G T G T A T G A G G C G - 3 ' . IL and IR flank the 2kb deletion in gklO. IC2 recognized a sequence located within the deletion and could generate a product with IR. Hence, two products could potentially be amplified in W T by the three primers: a 3052bp product generated by IL and IR, and a 595bp product generated by IC2 and IR. The 595bp product was amplified preferentially and the 3052bp product could not be detected. Since the recognition site for IC2 was missing in gklO, only one product of 1020bp could be produced. After worm lysis, lysate was used directly as template in PCR. A 25 uL reaction contained I X PCR buffer (50mM Tris pH8.3, 35uM N a H P 0 , 0.05% Tween-20), 2.0mM 2  4  M g C l , 0.4mM each of primers IL, IR and IC2, 0.2mM of each dNTP, and 1U of Taq 2  polymerase. Thermal cycling conditions were the following: initial denaturation at 95 °C for 4 min, 34 cycles of 95°C for 30 sec, 56°C for 30 sec, and 72°C for 1 min, followed by final elongation at 72°C for 10 min. PCR products were separated on 1% agarose gel and D N A was visualized by ethidium bromide. 2.2.4 Measurement of forward mutation rate in dog-1 (gklO) by the eTl translocation eTl as a mutagen test system  51  The eTl system was used to measure the potency of gklO as a mutator. eTl is a reciprocal translocation between chromosomes III and V , resulting in two aberrant chromosomes designated eTl (in) and eTl(V). In eTl(III), the left half of chromosome V (VL) is translocated onto the left half of chromosome III (IIIL). Whereas in eTl(V), the right half of chromosome V (VR) is translocated onto the right half of chromosome III (IIIR) (Rosenbluth and Baillie, 1981). In eTl heterozygotes, eTl(III) and eTl(V) pair and segregate from the normal chromosome III and chromosome V respectively. The consequence of this pairing behavior is that crossing over within V L and IIIR is essentially eliminated. The breakpoint on chromosome III disrupts the gene unc-36, resulting in an uncoordinated phenotype in homozygous eTl. eTl has been used as a mutagen test system to measure the frequency of recessive lethal mutations within the crossover-suppressed region on chromosomes III and V after treatment with mutagens or radiation (Rosenbluth et al., 1983). It is achieved by generating a strain heterozygous for eTl and normal chromosomes III or V . Chromosome V was marked by dpy-11 unc-42, which map within the crossover-suppressed region. In this case, a heterozygous eTl parent segregated wild-type, Unc-36, and Dpy-11 Unc-42 progeny. If a lethal mutation occurred within the crossover suppressed regions on the normal chromosomes III or V , Dpy-11 Unc-42 progeny would not survive, resulting in absence of this phenotypic class. A dog-1 dpy-11 unc-42 hermaphrodite (KR3837) was crossed to heterozygous eTl males. Phenotypically wild-type offspring from the cross segregated Wilds, Unc's and Dpy Unc's. From these offspring, Wild progeny were screened for gklO homozygosity by P C R with primers IL, IR and IC2. A single individual was used to generate the strain, whose genotype was dpy-11 unc-42/eTl; dog-l/+. The test strain was expanded for one generation after confirmation of gklO homozygosity. Subsequently, approximately 1350 Wilds Po hermaphrodites were set up on individual plates, and FI was screened for absence of DpyUnc's. Parents that gave rise to few progeny due to early death or sterility, and parents that segregated Dpy progeny were discarded. Dpy progeny could arise if unc-42 was outside but very close to the crossover-suppressed region. In such case, crossover between unc-42 and dpy-11 in the eTl heterozygous parent would generate a dpy-11 marked chromosome V and unc-42 marked eTl(V). A lethal event was verified when Dpy Unc's were also absent from F2. Approximately 1350 heterozygous eTl heterozygotes were set up individually and  52  screened for absence of Dpy-11 Unc-42 progeny, the frequency of which provided an estimation of the forward mutation rate. 2.2.5 Detection of deletions For each repeat, at least 16 dog-1 (gklO) and 16 N2 worms were screened for deletions. Single worms were lyzed as described in section 2.2.2, and the lysate was used directly in PCR. A 25pL reaction contained I X PCR buffer (50mM Tris pH8.3, 35uM N a H P 0 , 0.05% 2  4  Tween-20), M g C b to the indicated final concentration (Appendix A), 0.4mM of each primer as (Appendix A ) , 0.2mM of each dNTP, and 1U of Taq polymerase. Thermal cycling conditions were the following: initial denaturation at 95°C for 4 min, 34 cycles of 95°C for 30 sec, the indicated annealing temperature for 30 sec (Appendix A ) and 72°C for 1.5 min, followed by final elongation at 72°C for 10 min. PCR products were separated on 1% agarose gel. The gel was stained with SYBR® Green I nucleic acid stain (Molecular Probes) and scanned by a Molecular Dynamics Storm 860 Phospholmager system (Amersham Biosciences). Bands with smaller size than wild-type were sampled randomly and sequenced. Sequencing was carried out using Big dye Terminator cycle sequencing V2.0 on a 377XL D N A Sequencer (Applied Biosystems) at the N A P S facility at U B C (Vancouver, Canada). 2.2.6 R N A i silencing of dog-1 expression E. coli cells expressing the GenePair Fragment F33H2.1 obtained from the C. elegans Chromosome 1 R N A i library (purchased from Julie Ahringer, U K H G M P Resource Center) were grown in L B medium for 6-8 hours. About 20uL of cells were then seeded onto regular N G M plates spread with ampicillin, carbenicillin and IPTG to final concentrations of 50u.g/mL, 25pg/mL and ImM, respectively. Plates were incubated at 37°C overnight. Single N2 L4 larvae were grown on R N A i plates. After 2.5 days, eight L4 progeny from each parent were transferred to a fresh R N A i plate. D N A of the progeny was analyzed for deletions after two days.  53  2.3 Results 2.3.1 gklO is a deletion allele of dog-1 and confers a mutator phenotype The dog-1 gene (original sequence name is F33H2.1) encodes a predicted 983 amino acid DOG-1 protein (Figure 2-1) that contains seven protein motifs characteristic of the D E x H box type of D N A / R N A helicases. A B L A S T search showed that the predicted protein is most similar to human BACH1/BRIP1 (David L. Baillie, personal communication), which is a 5' - » 3' helicase that interacts directly with BRCA1 (Cantor et al., 2001; Cantor et al., 2004). Alignment of C. elegans DOG-1 and human BACH1/BRIP1 is shown in Figure 2-2. Within the C. elegans genome, the highest sequence similarity to DOG-1 is the gene product of F25H2.13 (Mike Schertzer, personal communication; see Figure 2-3 for alignment of DOG-1 and F25H2.13). The non-Caenorhabditis protein that has the highest score in a B L A S T search using F25 H2.13 is R T E L , a mouse helicase-like protein that is proposed to contribute to telomere length regulation in mouse species (Ding et al., 2004; Zhu et al., 1998).  Figure 2-1. Intron-exon organization of dog-1 (F33H2.1) and its gklO-xmll allele. The gene contains 14 exons encoding 7 conserved motifs of D N A / R N A helicases, encompassing a conserved DEXDc2 domain of the DEAH/DEAD-like helicase within superfamily 2 and a HELICc2 carboxy-terminal domain of the helicase superfamily. The deletion in gklO removes exons 2-8 and introduces a premature stop codon in exon 9 (Vijayaratnam, 2000). (Figure adapted from Cheung et al., 2002)  54  K&K3HTR3 A F Q S V K E E QP 3T 3§E P D B K S P J H H E IAgEH IKS? lUQ|KJIDEYE| 3 QLAHfflTS ILRG-JHSKQHCLLElpT&SGgSgALLCSgL A W Q | SSL : SH3P ADE GS3 EK AE'  D O G 1*0 BACHlxl  JJS33D MSStraSEYTNGGVKI;  D0G_lx0  MHLGUP VRUPRGLSLY3TQKLKIVRILy ALKN3Q.BlTLGEBPT&B&KTHAJJLBST cfJ^KQVJlDEKRE3  B ACH 1x1  SC C C ACH3KD FTBHBHH Q GTSPJtrHYPgTPP 3 ERjyGT S3 |c QDgPEKTTgAgKL SgKKq ASyYRDENDD FQV^KRggPLETTQ IT K B i B I P T E S K V Y E E P P E E E ErfJEPvJgKHDiaRE A ^ K D r T P ^ P & E ^ P V T L J ^ L E P j g S p E P U E J g c T CL?  DOG_lxO BACH i x l  DOG_lxO BACH 1*1  KJJSCEBJJGFSGKT  :c rgrESmn.ri A K U D S GKT V K J H - S ^ E K  iKsgs P0KPP G H C  3gc C C SJJKQ GHS QsJ SS B—T K E P H T G K M S I — g - - .  •laW G T P . T K K Q IAQJJ Lfgr s | L P | A K | | L K H ^ A g g Q g g M A A R K H AD I3QYgKggNSJg3 I GCSgKS AHKP|rEKALPjPJ)HL / T G T R T H K Q I Ay I : WGN r N R H E K g i g a L D g g H & K S c|FYHGVHg 13D Q H T | Q T FQ II  DOG_lxO BACH 1x1  DOG_lxO BACH I x l  - - - | - - | C K AWD|E3L1S|GKKLK  ACPfsgAgEg I  O f AJ™3J  ASSBE I D K T G I E E S L L J I3D S E S H L K EI FRGHLL YL gH JjLEHL RWIRQSSSE AKTP ARGGQDj z f J p t p n O r - - IR^jgEJjLgAjcCSL, I1TWLE AN AaYflEERD YE 3 ACKCWBGIIEMLI. TL! FASDEj  iFaSKEftpD 3 I L S L R I K j i  J<Mk  D O G 1x0 BACH I x l  |QSTY 3c[3l33AHL IT 3LTDPE HGUDL FT PPLTPKHA A S A C S ^ H J T I S N S P E H Q V R D A rKP 3 3T A ^ C IEKwSVFQ 3 YH&N Q QXQ 3AJJFP IBOHHHBHJL QfiEgK ISP IgGKEE A R E y p 3 j 3 A S T Q Q H L K G L Q K V L D Q L  D0G_lx0 BACH 1x1  D|3 RL H13 IEP I HQ GgHHTgD AD < H & B J G S P RPTTTRS 3 A G P R S « Q Y K S E W W J A D AASD GDDwjDP 3 M S E | & H | P I3EGCKT AfjQ MUD I S!KS?SL 3VLP S»K- -KRQRQ" TAVH  DOG_lxO BACH Ixl  Tfj3L2H£ iEl 5i3ArHET: jSgiMGKWQi  DOG_lxO BACH Ixl  SSBYCjj  DOG_lxO BACK 1x1  DOG_lxO BACH 1x1  FR-  QSvBaTS  s  FEHQDi rags  KD  3  SN SiffiQT  • m-  • ru-  JQCMRRMSTI  SARVS  .3 3 E1T SVHfflQ FD AA i m p S | QGGEKTN—FfflELL QUv|nA Ij£  H H I T <l A Y R A L U Q A L GP.CLRH O S M E I CI AYR ALHQ A L GRC I I H  AH  : GKVSE GIB l&KVSE GOD |8j LERSTGML nsiigpsRvEsl  QCWgR^SDPE GS  ;rs  3 Y P 5 R K E F H A H F R B H I Q J3JSK A3E K A K K E N F C E A J H S I ^ Q K S L H V S IKDRTNIODME STLEUTSL  IQHSQHHS^SALESL  DOG 1x0 BACH 1x1  KYSTPPYLLEAA3HL3PENFVEDE AKICVQELQCPKIITKN3PLP3S 113RKEKHDPUn.EE AGKAEKIUISRST3PTFOTCQTK  DOG_lxO BACHlxl  RV 3103 3 FN3 L GQ VFT GKIPK ATPE LGSSEKSAS 3PPRFKTEKHE 3KT VLP FTDK CE 3 3NLTUKT SFGSCPCISETIISSLKIBAT  D0G_lxO BACHlxl  LTRKHH3EKPLC SEE AL DFDIEL 3LUSEEDKQ 3T3BRDFETEAEDE3 IYFTPELYIiPEDTDEEKTOL AETDRGHRL AHHSDC IL  DOG 1x0 BACHlxl  AKDLFEIRT IKEVDSAREUKAEDC IDTKLHGILHIEESK IDD ID GHVKTTW IDE LEE GKTHE IE IKNFKP 3P SKHKGMFP GFK  Figure 2-2. Alignment of C. elegans DOG-1 and human BACH1/BRIP1. The seven conserved helicase motifs are indicated by Roman letters above the sequence. Sequences were aligned by Clustalw at NPS@ (http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html).  55  F33H2.1 F 2 S H 2 . 13  jSSSDArTOMFAimffiGKSimiSArQUUm^ J p PK ASffST EVWIMP^L 3UKIYFEP"2EC<JRI rHKHWigULa^LD AAL  F33H2.1  SLYSTQKLK IVRILTALKSSQHVL ( £ ^ Q S J S 3 K A ^ A | J J Q C Q % C Q  F2-5H2.13 F33H£ . 1 F25H2.13  F3 3 H Z . 1 r2SH2.13  ^  Es^C^B  Q R Q K  VIJE3RE  i is r  SKEKCE I H B F S R K T Q WFTHD iglPLESKVYEEP  P L D r M W Q T 3  IS ^ a  M K T D E E L K S  B  ? U I  '  T  PEEEEFIEPUPUKHMX FE AQWEHDFTPHTPOETPPUTLEEELEPUKKPEPVET KCTCLPRVRJJJ jjSK» A^M-<aF3gLP B J . i B t B . s B f c S B B T B L H i ^ r El  i g g H ^ f f l g Q SHEJP AAygKAjJj I3QYCJ^EVNSAH§I & C 3 FKSAKKPRFEKAL PLRDKLE P O f e v | p ^ S | / E T L A13 VP QJj * ® r B EHFEQKwSlKgSimQA^CRGLl^KRACir^  F33H2.1  S<3MRILTCDASfHFc3jjtegIjfflL{BursSD|QQ  jjnLJjRj E j g p T B R E A B U r  F25H2.13  «^QR3ETA.3^LJE»HQtSFS* ^12  W l r B R B S f E l s IBE BgEBAELT ST S I AL C IEE LKKtiL AL LVDEEET A R S E A  v  s rr|KELiifojjngjte.i gpjKAg|rgjBBri>  F33H2.1  BAET&AF&3 A K I D L 7 K K L l S ^ R T ^ | S B  F33H2.1  KT P AR&&Q D dVSSI CT 13 S R N L F T 3 JrSPEHGSJDL F T P P L T P K H A C V A AS F 3 A I T K H H E P E M Q V F D A § K P 3 S T A I V C H E K O T . J | F C ! 5 Y F &  F2SH2.13  B A I S Y L L 3 K H E E V A L T EKgB G M E K J A 3 F L L S { J V 5 T H A Q D UAAAUgEE T U K L U D R U D P I C T U A R 1 T C K L * IQ KDKDHEKflT I K V Q C F Q A 3 I  F33H2 . 1 r25H2.13  HQ QYQSTYRJHI3 IEP IHQM&RFJtHTTOADU3K3T3FSHPRPTTTRS 3 A&PRKMQYKEEHAUL AD AAAD GDDWKDP3K3ET&HKP ISE SHRHLKMR&I  F33H2.1  OCKTT I3LMCW3P ALSrn) ArHETg3fJ2  T25H2.13  SpSS  L  E  B  I  R  3  3 S  E  S S '  I E EK^itf^Ri:v?iLKgrR&ajgLV»E5L I L J H . R U I J Q S S T E A  F2SH2.13  R  (  '  S  S Q I K S ^ L S & ' A S J & E ILiJrr|AjA&r2AlTSVE J . J D V L R  LKTEJjJBTE K K Q Qj^'QUflwOm RrAAjjfe ifiprBHRI Q C T Y R K T SIPPE  CTcfrfr)T  1 1 1 3 1 ^ 0 A FTVW|B^itB * &  ?HAlKQUP 8B.T 3 ™ J T R K K P J G L A63 FOHRKKLDY  -IV-  F25H2.13  s|rYc5L&A I I K Y S C S H I S A E Q C R L P ^ R V 3 Q Q % C Q C M I u3&UAgALLR. . . JmugQBJgiBrsHsQlHl^ATWKTTK^  F33H2.1  V FgAjriH&sLHFAifraB  ^^^JMsWSSS^iiMQSsi^SaSms-KE^  F25H2.13  AJJLLAVC  ^cSjAE SgAjjj 103  F33H2 . 1  3 H 2 .1 F2SH2.13  lj£  CajTOSH^gA^igEaLERQT J U S plflKrlarWirlYASAKPE SIT  RHgTHROlgDcSuffivi^raRS S^rSJ&ID Q&PAgRrJjPSR  ILSCD  SF  T  Q  VII^I&3  H F '"B-^W-^ffTf-yLM&RgD TKpERQ 3 3 Q^jjfiQtgPJg  AS 3 ARVSJj^gAQlKSYPgjlFKE FN AN r % B I QSRSAgKAJfcMTrCE S F H H T ' S R S D W D & C ALKT s3FRKEl&fflL@HsSr5v IKKQ AKQ CKS FR QVKQ  F33H2.1  F2.5H2.13  F33H2.1 F2 SH 2 . 13  TAASDSKDD I IE ITLEDHFSP ASHKLEKKE IT KLRPPQL  IPSTSSUFSLPTHEDELKIKKMEOEHDIQCLSSIPLESHKRKrKIETPG  PSTSTLTQK 3EPPKKKK ILLLTRHTLPDE YQK A IE IPT 3 ELLKDHSD DNKKQ FATTLRSYKAE 3 IRWD E VFQRFRP IFUPHKAD L FIA  F33H2.1  F2«H2.13  CSMVLR3EDJMKYLKKALE5KIHT  Figure 2 - 3 . Alignment of C. elegans DOG-1 and F 2 5 H 2 . 1 3 gene product. The seven conserved helicase motifs are indicated by Roman letters above the sequence. Sequences were aligned by D I A L I G N (http://bibiserv.techfak.uni-bielefeld.de/dialign).  56  The dog-1 allele gklO (generated by the C. elegans Gene Knockout Laboratory at the University of British Columbia, Vancouver, Canada) carries a 2032bp deletion which removes exon 2 to exon 8 and introduces a premature stop codon in exon 9 (Vijayaratnam, 2000; Appendix B). The resulting protein would at most consist of one of the seven helicase  dog-l(gklO) F25 F26 F27 F28 F3Q F31 F32 F33 F34 F35 F36 F37 F38 F39 F40 F41 F42 F43 F44 F45 F46 F47 F48 F49 F50 F51 F52 F53 F54  brood size  it it  (vab)  1-50  •  50-100  •  >100  •  (mil)  f-CJp <tet)  Figure 2-4. Variable brood size and mutator phenotype in dog-l(gklO). Generations F 2 5 F54 nematodes were followed by transferring single animals. Brood size is indicated by size of circle. Horizontal lines indicate analysis of siblings in a particular generation; V indicates extinction of the line. Heritable mutations are shown in boxes; the corresponding phenotypes were: mlt, molt defect; vab, variable abnormal; dpy, dumpy; lon/clr, long/clear; him, high incidence of males; let, lethal. Notably, the brood size is markedly variable and the frequency of heritable mutations is high relative to control nematodes (Bristol N2, right). (Figure adapted from Cheung et al., 2002)  57  motifs in a 195 amino acid truncated product. It is expected such deletion would result in complete loss of function. Hence, gklO is likely to be a null allele (Vijayaratnam, 2000). dogl(gklO) displayed a variable reduction in brood size, compared to the expected number of approximately 300 from a wild-type parent. Spontaneous mutants with characteristic phenotypes were recovered frequently in dog-l(gkl0) (Figure 2-4). To estimate the forward mutation rate in dog-1 (gklO), a mutagen test system using the reciprocal translocation eTl (III; V) was employed (Rosenbluth et al., 1983). The test system captures lethal mutations that occur within two defined regions in the genome: IIIR and V L . Such lethal mutations appear as absence of Dpy Unc's in the progeny. Therefore, the gklO allele was introduced into the test strain, and approximately 1350 Po worms were set up for screening of lethal events, as indicated by absence of Dpy Unc's. Dpy Unc's were absent in 16 out of 1336 plates. Therefore, the frequency of forward recessive lethal mutations within IIIR and V L was 1.2% in dog-1 (gklO). This was significantly higher than the spontaneous frequency of 0.06% measured in a wild-type background by Rosenbluth et al. (1983). The potency of gklO as a mutator is roughly equivalent to a 5mM E M S treatment for 4 hours, or a 5x 10" Roentgen dose of gamma radiation (Rosenbluth et al., 1983). 4  2.3.2 Frequent deletion of a (G/C) within vab-1 in dog-l(gklO) 20  Among the different spontaneous mutants derived from dog-1 (gklO), three had the same Vab phenotype that was characterized by a "notched" head and reduced penetrance. They were isolated independently and all mapped by complementation to vab-1 (III), a gene that has been cloned and identified as a tyrosine kinase (George et al., 1998). To understand the molecular basis of the three vab-1 mutations, primers were designed to amplify the different exons of vab-1. Strikingly, all three mutations were deletions initiating in the same run of 20 continuous cytidines just outside exon 5 of vab-1 (Figure 2-5). The second breakpoint occurred at various locations. Individual nematodes with no apparent Vab phenotype were also screened for de novo vab-1 deletions. In approximately 1 of 10 dog-1 (gklO) worms, a lower molecular weight band was observed in addition to the expected wild-type band of 500 bp (Figure 2-6). The bands were confirmed to represent de novo deletions by sequencing (Figure 2-5). Similar to the three vab-1 exon 5 germline deletions, all the somatic cell deletions had one breakpoint located just before or within the (C) tract. Hence, dog-1 (gklO) 20  was generating deletions of the (G/C) upstream of exon 5 of vab-1. 20  58  A  B 100  DEL tgaaaagtatt-tctaacgaaaooccccccx'ccccccx-ciccct'atcagcctaac-atgcct tgaaaagtatttctaacgaaacc tgaasagtatttctaac tgaaaagtatttctaacgaaaceee  1  (QJO  200  300  '  400 I  I  vab-1 axon 5  334 g 130 g 97 g  tgaaaagtatttctaacgaaa.  392  tgaaaagtatttctaacgaaaccccca tgaaaagtatttctaacgaaaecac tgaaaagtatttct-aacgaaacccco tgaaaagtatttctaacgaaaccec  384 382 245 201  tgaaaagtatttcfcaacgaaaaaa. tgaaaagtatttctaacgaaacco tgaaaagtatttctaacgaaa  183 172 128  Figure 2-5. Structure of germline and somatic deletions involving vab-1 exon5 in dog-l(gklO). Top, nucleotides flanking the (C) just outside exon 5 of vab-1 (intron-exon boundary is 51 nucleotides away from end of the repeat tract) in which deletions were found to be initiated (grey box in C). A) Deletion position relative to the C- tract. B) Size of the deleted (del) fragments (g, germline deletions). C) Breakpoints relative to exon 5 of vab-1. Inserted sequences are underlined in A . (Figure adapted from  500 I  5kb4kb3kh-  g -mm «Aw m •J• •<ippi - iw^^-fl^^^p-^w^w i ^ ^ w m M M  J  H  M  M  M R  f H |  am  W  « g »  <  M  JH^W '^RPJV' ^IBPlBr 'flPHiP' W^Bf -<nlnSP  ;  mm,  2kb-iW  1  Figure2- 6. Vab-1 exon5 was involved in deletion at a high frequency in dog-1 (gklO). This i a representative gel showing the presence of deletions in 2 out of 18 individual dog-1(gklO) worms. The deletion bands are marked by the asterisks.  2.3.3 Frequent deletion of a (G/C) tract within the cosmid F55F3 in dog-l(gklO) 26  The cosmid F55F3 of chromosome X also contains a (G/C)n in an intergenic region. To determine i f this (G/C) was also involved in frequent deletions in dog-1 (gklO), primers 26  were designed to amplify a lkb region around the tract. PCR products with lower molecular weight than the expected wild-type product were readily amplified in dog-1 (gklO) (Figure 27). Multiple bands could be amplified in almost all dog-1(gklO) worms. Sequencing of bands sampled randomly confirmed that they were de novo deletions (Figure 2-8). Similar to the deletions in vab-1, the deletions in F55F3 initiated at the 5' end of the (C) and extended for 20  various lengths (Figure 2-8). In a rescued strain where a wild-type copy of the dog-1 gene was introduced back into the Dog-1 mutant, the frequency of deletions was significantly reduced (Figure 2-7). A plot of deletion frequency against deletion size showed that deletions of 100-200bp were the most common (Figure 2-9). The longer the deletion, the less frequent it was. Deletions of more than 500bp could be detected using primers that amplified a larger fragment (data not shown). Deletions could also be observed in heterozygous progeny from a cross between dog-1 (gklO) hermaphrodite and N2 males (Figure 2-8). Deletions in heterozygous progeny could be a result of haploinsufficiency, maternal effect, or segregation of phenotypes in the parental germline.  60  dog-l(gklO)  1  4  Rescued  4  1  kb  1.5  10  •v • •  0.5  Figure 2-7. Disruption of dog-1 leads to frequent deletions involving the (G/C) in F55F3. One or four dog-l(gkl0) or transgenic dog-l(gkl0) worms carrying full-length dog-1, indicated as "Rescued", were lyzed in a single lysis reaction. Each lysate was divided and two independent PCR reactions were carried out to amplify the F55F3 fragment. Expression of wild-type dog-1 rescues the mutator phenotype of (dog-l)gklO. The wildtype fragment is 1061bp in length. (Adapted from Cheung et al., 2002) 26  2.3.4 Somatic tissues are susceptible to deletions in dog-1(gklO) The fact that heritable mutations arose in dog-1 (gklO) (e.g. vab-1) demonstrates that deletions occur in the germline. To test if somatic tissues are also susceptible to deletion, dog-1 (gklO) glp-4(bn2ts) was generated. Glp-4 is required for development of the germline (Beanan and Strome, 1992). bn2ts is a temperature sensitive allele of glp-4. A t the permissive temperature (14°C), worms are fertile but have reduced progeny size. A t the restrictive temperature (25°C), the mutant develops much fewer germ nuclei. These germ nuclei fail to enter meiosis and the mutant is essentially without a germline. When dog-1(gklO) glp4(bn2ts) grown at 25°C or 14 °C was analyzed for deletions in F55F3, it was found that those grown at 25 °C had deletions at a frequency comparable to those grown at 14°C (Figure 211). Therefore, deletions occur in both the germline and somatic tissues in dog-l(gklO).  61  A  B C 400 DEL  C4ACKTCTC<  gacgacgattcgacgcaaacoccacocaccccooccccccoocoocoocctctcccctcatttcaccaat gacgacgattcgacgcaa gacgacgattcgacgcaaacccca gacgacgattcgacgcaaaccccaccccc  an F  700 1  eoo 1  son 1  1000  -H  377 377 3B4  gacgacgattcgacgcaaaccGcaccc  tsj  500  •—'g 1 — ,  344  gacgacgattcgacgcaaaccccfcactgaa.cca.tgfla^aaafrnfl gacgacgattcgacgcaaacccc gacgacgattcgacgcaaa gacgacgattcgacgcaaaccccacaagajsas gacgacgattcgacgcaaaccccaccccccc gacgacgattcgacgcaaacccc gacgacgattcgacgcaaaccccacc  339 334 249 134 132 109 80  Figure 2-8. Structure of deletions involving the (G/C) in an intergenic region within the cosmid F55F3. Nucleotides flanking the intergenic sequence C4AC26TCTC4 (grey box in C) present on the cosmid F55F3, and breakpoints relative to this sequence are shown in A and C. The size of sequenced deletions is shown in B. Inserted sequences are underlined in A . (Figure adapted from Cheung et al, 2002) 26  •  ^  i  deletion  ^-771  r  1  size (bp)  Figure 2-9. Size distribution of deletions involving F55F3 in dog-l(gklO). Deletions of 100-200 nt were observed most frequently. (Adapted from Cheung et al., 2002)  2.3.5 R N A i silencing of dog-1 expression R N A interference (RNAi) is a phenomenon in which gene expression is reduced or eliminated by targeted degradation of mRNA by dsRNA, such as a small interfering R N A (siRNA). To further demonstrate that the deletion phenotype in dog-1 (gklO) was indeed due to the disruption of dog-1, R N A i by feeding E. coli containing plasmids producing dsRNA for dog-1 (Kamath et al., 2001) was carried out in N2 worms, which were then analyzed for deletions in F55F3. N2 was fed with the E. coli strain expressing the GenePair Fragment F33H2.1 obtained from the C. elegans Chromosome 1 R N A i library (purchased from Julie Ahringer, U K H G M P Resource Center). Deletions in F55F3 were observed in worms from the first generation after R N A i treatment was initiated (Figure 2-12). Deletion frequency varied from one brood to another, which could be due to differences in the efficiency of RNAi.  63  kb  mmm -1 O  -0.8 -0.6  Figure 2-10. Deletions in F55F3 in dog-1 heterozygotes. Dog-1 (gklO) hermaphrodites were crossed to N2 males. Heterozygous hermaphrodite (A) and male (B-C) progeny were lysed and lysates were used in PCR amplification of the repeat tract in F55F3. Each lane contains PCR products amplified from a single worm. The wildtype fragment is 1061 bp in length. A and B were progeny from the same parent.  64  Figure 2-11. Somatic tissues are susceptible to deletions in dog-l(gklO). Dog-l(gklO) glp4(bn2ts) grown at (A) the permissive temperature of 14°C (worms developed the germline) or (B) the restrictive temperature of 25°C (worms did not develop the germline) were analyzed for deletions in F55F3. Each lane was amplified from the lysate of a single worm. The wildtype fragment has a length of 1061 bp  2.3.6 Genome-wide deletions in dog-l(gklO) The high frequency of deletions involving the (G/C)n tracts in vab-1 and F55F3 suggested that dog-1 was required for the maintenance of such repeat sequences. To see i f instability of (G/C)n were a global phenomenon in dog-1 (gklO), nineteen additional randomly selected (G/C)n were analyzed for deletions in the mutant strain. Deletions were observed in roughly half of the tracts (Table 2-1). No deletions were detected in any of the five tracts with 18 or fewer guanine nucleotides. To assess whether deletions in a\og-\(gkl0) were a general phenomenon affecting all types of repeats other than (G/C)n, other types of repeat sequences including three (A/T)n, one (AT/TA)n, two (CT/GA)n, two (CA/GT)n, four (CTG/CAG)n and nine (CCG/CGG)n tracts  65  were analyzed by PCR (Appendix C). No deletions were found in any of these sequences, suggesting that deletions are restricted to (G/C)n in the mutant.  B  •tM mt ^  mm  w  4 .'  mi  Figure 2-12. Inhibition of dog-1 expression using R N A i resulted in deletions similar to dogl(gklO). N2 worms were fed with E. coli expressing the GenePair Fragment F33H2.1 for two generations. Worms from the 2nd generation were analyzed for deletions in F55F3. Shown here are worms from 3 different parents (A-C). Each lane was amplified from the lysate of a single worm. (Adapted form Cheung et al., 2002)  2.3.7 A proposed model for deletions of (G/C)n in dog-l(gklO) Because deletions in dog-1 (gklO) seem to initiate at the 3' end of (C)n tracts and to involve about half of the tracts examined, it was proposed that these deletions result from failure to resolve secondary structures of guanine-rich D N A that may arise sporadically during lagging-strand D N A synthesis (Cheung et al., 2002). A mechanism dependent on length and orientation and suggestive of in vivo lagging-strand secondary structures has been proposed to explain the instability of trinucleotide repeats in Escherichia coli (Hirst and White, 1998; Kang et al., 1995a; Trinh and Sinden, 1991) and Saccharomyces cerevisiae (Balakumaran et al., 2000; Rolfsmeier et al., 2001). On the basis of the specificity of the deletion phenotype involving (G/C)n in dog-1(gklO), and of the highly conserved helicase motifs in DOG-1, it was proposed that DOG-1 is required to unwind secondary structures of guanine-rich D N A (Cheung et al., 2002). Formation of secondary structures, including hairpins and G quadruplexes, has been documented in vitro in numerous studies, and it is widely believed that such structures also occur in vivo and that they have implications to many cellular processes (reviewed in Simonsson, 1998).  66  Table 2-1. Frequency and size dependence of deletions involving (G/C)n in dog-1(gklO). (Adapted from Cheung et al., 2002) Location  Chr  Tract length  Y77E11A Y75B7AL Y39A3CR.6 Y41E3 ZC123.3-a ZC123.3-b M03A1.1 M01E10.2 C04C11 Y15E3A F42C5 R03G5 F46H6/C07A12 Y41D4A C18F3 R144 R11B5 F55F3 F49E10 F38A6 B0524.1  IV V III IV I I II III X X IV X X IV IV III X X X V III  G14  G,  5  G5TG12 C17  G18 C19 C20 C20 C20 C20 C1 2  C22 C22 G24  G AG C C 2  2 5  2 5  2 5  C26  G26 C29 C32  67  Presence of deletions  Estimated deletion alleles per adult  no no no no no yes yes no no no yes no yes yes yes yes yes yes no yes no  0 0 0 0 0 0.2 0.1 0 0 0 0.4 0 multiple multiple multiple 0.5 multiple multiple 0 multiple 0  2.4 Discussion 2.4.1 DOG-1 and its homologue in human The gene dog-1 encodes a putative DExH-box helicase of superfamily 2 based on sequence homology. Proteins in this superfamily include both R N A and D N A helicases and are present in both prokaryotes and eukaryotes (reviewed in Hall and Matson, 1999; Gorbalenya and Koonin, 1993). However, biochemical evidence for DOG-1 being a helicase is lacking at this stage. To prove that DOG-1 is indeed a helicase as suggested by its sequence will require in vitro demonstration that the protein catalyzes the unwinding of nucleic acid strands in an ATP-dependent manner. A B L A S T search showed that besides the C. briggsae homolog of DOG-1, the human helicase BACH1/BRIP1 is the most similar protein to DOG-1 (David L. Baillie, personal communication). BACH1/BRIP1 binds to the B R C T domain of B R C A 1 , and this interaction is required for the cell-cycle checkpoint function of BRCA1 (Cantor et al., 2004; Cantor et al., 2001; Y u et al., 2003). Recently, the C. elegans homologue of B R C A 1 , BRC-1, has been identified (Boulton et al., 2004). Ce-brc-1 (RNAi) displays an elevated frequency of chromosomal nondisjuction under normal conditions, and upon treatment with gammaradiation, exhibits increased germ-cell death and chromosomal fragmentation when checkpoint function is intact (Boulton et al., 2004). Phosphorylation of human BACH1 at Ser990, and the presence of several other conserved residues located at the C-terminal are critical for binding to B R C A 1 (Clapperton et al., 2004; Shiozaki et al., 2004; Y u et al., 2003). DOG-1 lacks this C-terminal domain of BACH-1 (Figure 2-2). Therefore, it raises the question of whether DOG-1 and BRC-1 interact directly, and i f they do, how. The two proteins may interact indirectly via some adaptor protein, but it is also possible that they do not interact in vivo. C. elegans proteins with the highest scores in a B L A S T search using the C-terminal domain of BACH1/BRIP1 include immunoglobulin-like proteins, structural proteins, and proteins involved in D N A metabolism and cell cycle checkpoints (data not shown). No single C. elegans protein shows a particularly high level of similarity to the C-terminal domain. Therefore, any functional interaction between DOG-1 and BRC-1 might be very differenct from the human counterpart. It is noteworthy that BRC-1 is substantially smaller than the human counterpart (596a.a. versus 1863a.a.) because sequences resembling BRCA1 exonl 1 are missing in C.  68  elegans brc-1. Therefore, BRC-1 may not perform all the functions of B R C A 1 (Boulton et al., 2004). In such a case, the D N A repair pathway involving B R C A 1 in humans and BRC-1 in C. elegans may have diverged. Alternatively, the two functionally related proteins might have evolved independently in the two species. 2.4.2 Deletions in dog-l(gklO) rarely resulted in heritable mutations Although certain (G/C)n tracts had a high frequency of deletions in dog-1 (gklO), heritable deletion events were extremely rare. A deletion allele of a (G/C)n would not be passed onto the progeny i f its integrity and/or flanking sequences were essential for development or i f caused embryonic lethality. This could explain the absence of heritable deletions in (G/C)n that are intragenic (e.g. the tracts within C18F3 and F38A6) or that are close to genes (e.g. the tract within F42C5 is 510bp away from a downtream gene and deletions may affect the promoter region and the beginning of the gene). However, there are examples where lethality could not explain the absence of heritable deletions. For example, the tract within F55F3 is located in an intergenic region and is 8kb away from the closest gene. Neither of the two flanking genes appears to be essential: F55F3.3 encodes a protein predicted to display a sodium/potassium ion ATPase activity, and R N A i resulted in no visible phenotype (Kamath et al., 2003); F55F3.t encodes a fRNA-Lys, and this gene can be found elsewhere in the genome. Therefore, it is unlikely that deletions of the (G/C) in F55F3 were lethal. This 26  particular (G/C) deleted at a high frequency in dog-1 (gklO) animals, yet no heritable 26  deletions have been observed. Another possible explanation of how heritable deletions occur in such a low frequency is that deletions happened mostly in somatic tissues. Deletions detected by the P C R assay described in this chapter could come from the germline or somatic tissues. Heritable deletions involving a (G/C)n (within vab-1) were observed, demonstrating that deletions did occur in the germline. To find out i f deletions also occurred in the soma, dogl(gklO) glp-4(qm37) double mutant was generated. Glp-4 (qm37) grown at restrictive temperature (25°C) develops only a few nuclei in the gonad and is essentially germline-less (Beanan and Strome, 1992). Deletion analysis showed that the double mutant grown at 25°C carried F55F3 deletions at a frequency comparable to that grown at 14°C (Figure 2-11). Therefore, both somatic tissues and the germline were susceptible to deletions in dogl(gklO).  69  If deletions happened in somatic tissues, how would the germline be protected? It is plausible that another protein assisted in maintaining the tracts in the germline when dog-1 was mutated. The most similar C. elegans protein to DOG-1 is encoded by F25H2.13 on chromosome I (See figure 2-3 for alignment of DOG-1 and F25H2.13 gene product). A B L A S T search shows that the F25H2.13 gene product is the most similar to human N H L and its mouse homologue RTEL. It has been demonstrated that R T E L is a dominant regulator of telomere length in mouse, and it is required for the integrity of telomeric D N A (Ding et al., 2004). Differentiating RTEL-/- embryonic stem (ES) cells display chromosomal abnormalities that are suggestive of a potential role of RTEL in preventing illegitimate recombination between telomeric repeats and internal repeat sequences (Ding et al., 2004). Interestingly, F25H2.13 R N A i on N2 appeared to alter recombination frequencies (Appendix D), although no significant difference in telomere length was observed (data not shown). RTEL-/- ES cells show no instability of (G/C)n; furthermore, F25H2.13 R N A i on N2 or dogl(gklO) did not significantly increase the deletion frequency of the (G/C) in F55F3 (data 26  not shown). Such findings argue against F25H2.13 encoding a protein that could prevent germline deletions in dog-l(gklO). A n enhancer screen could uncover genetic factors that might contribute to protection from deletions in the germline. 2.4.3 Deletions of different (G/C)n occurred at different frequency In dog-1 (gklO), deletion frequency differed among the different (G/C)n tracts examined (Table 2-1). In general, deletion frequency was higher in longer tracts, indicating involvement of secondary structure formation. A n exception was the (G/C) in R144, which 25  is relatively long but whose deletion frequency was comparable to shorter tracts. The question that arises is what determines the deletion frequency other than possibly tract length? Based on the model of secondary structure formation during lagging strand synthesis, Cheung et al. (2002) proposed that different tracts could have different deletion frequency i f the direction of the replication fork going through the tract changed during development. In this case, a tract whose G-strand was switched from being replicated by lagging strand synthesis to leading strand synthesis early in development, or from being replicated by leading strand synthesis to lagging strand synthesis late in development would have a lower frequency of deletion (Cheung et al., 2002). Switches in replication fork direction have been documented in Schizosaccharomyces pombe (Dalgaard and Klar, 2001). Furthermore, in  70  Drosophila and Xenopus, replication initiation occurs in random sites early in development but becomes confined at more specific locations later (Hyrien et al., 1995; Sasaki et al., 1999). Such reduction of the number of replication initiation sites during development would lead to changes in replication fork direction in certain regions in the genome. 2.4.4 The model Two forms of microsatellite instability have been extensively studied: one is the relatively small expansion or contraction of repeat tracts, the other is the rapid expansion of trinucleotide repeats. It is well established that defects in the mismatch repair pathway could lead to small changes in tract length because insertion/deletion loops cannot be repaired after polymerase slippage and strand misalignment during D N A replication (Liu et al., 1995; Ripley, 1990; Schofield and Hsieh, 2003; Strand et al., 1993). The mechanism of trinucleotide repeat expansion, on the other hand, is still speculative, but it has been proposed to be recombination-based and driven by formation of higher-ordered D N A structure (Richard and Paques, 2000; Wells, 1996). Deletions of (G/C)n observed in dog-1 (gklO) represents a novel form of instability involving microsatellite repeats that is distinct from the two forms of microsatellite instability mentioned above and is likely to arise by some different mechanism. The replication-based model proposed by Cheung et al. (2002) (Figure 2-13) could explain the directionality of the deletions: in all cases deletion begins at the 3' end of the (G)n (or the 5' end of the complementary (C)n). Firstly, only (G/C)n were affected in dogl(gkl0). Long (G)n may represent a unique challenge to the cell by forming secondary structures on the lagging strand that lead to a stalled replication fork. Alternatively, protein binding to the tracts could cause the blockage. However, this hypothesis is not supported by the fact that only approximately half of the (G/C)n examined were involved in deletions. If problem in dog-1 (gklO) was caused by protein binding to (G/C)n during replication, deletions in both directions should be observed, because whether the G-strand is on the lagging or leading strand would likely be irrelevant in such case. (CCG)n trinucleotide repeats cloned into E. coli were demonstrated to be unstable when the G-rich strand is on the lagging strand (Hirst and White, 1998). In such orientation, long repeats are involved in frequent deletions which in almost all cases are confined within the repeat tract. Hirst and White (1998) proposed that instability of the cloned (CCG)n  71  repeats are induced by secondary structures formed on the lagging strand. The difference in the structure of deletions involving (G/C)n in dog-l(gklO) from that involving (CCG)n trinucleotide repeats cloned into E. coli could be due to difference in the secondary structures formed and/or some intrinsic difference in the replication machinery between eukaryotes and prokaryotes. For instance, (CGG)n may form hairpin structures while (G)n may form G quadruplexes more readily in vivo; in addition, the size of an Okazaki fragment is 100-150nt in eukaryotes, much shorter than the l-2kb fragment found in prokaryotes (Benkovic et al., 2001; MacNeill, 2001). Such distinctions between the two systems could affect how  Figure 2-13. The invariable occurrence of breakpoints at the 5' end of the (C)n (i.e. the 3'end of the (G)n) is compatible with deletions resulting from the failure of lagging-strand D N A synthesis. According to this model, problem arises when the replication fork travels in a direction such that the G-strand is on the lagging strand (A-C). Single-stranded (G)n sequences used as a template for lagging-strand D N A synthesis are postulated to give rise occasionally to stable secondary structures (C, arrow). We propose that resolution of such secondary structures requires specialized D N A replication or repair machinery involving DOG-1. Failure to resolve secondary structures of G-rich D N A results in deletions by a mechanism that is unknown but is likely to involve excision and recombination, trans-lesion synthesis, or both (D). (Figure adapted from Cheung et al., 2002)  72  deletions occur. Therefore, the difference in deletion structure does not exclude the possibility that secondary structure formation during lagging strand synthesis may be the common cause of repeat instability in both settings. 2.4.5 Do (G/C)n tracts have any function in C. elegans"? Between the two types of mononucleotide repeats, (A/T)n tracts vastly outnumber (G/C)n tracts in almost all eukaryotic genomes studied (Dechering et al., 1998; Denver et al., 2004a; Toth et al., 2000). This bias may be related to the higher mutation frequency in (G/C)n compared to (A/T)n (Denver et al., 2004a; Gragg et a l , 2002; Wierdl et al., 1997): for example, in C. elegans, mutation rates of (A/T)n of 9 to 15 nt and (G/C)n of 8 to 16 nt were calculated to be 4.5 x 10" ± 3.2 x 10~ /generation and 9.0 x 10~ ± 1.7 x 10~ /generation 6  6  5  5  respectively (Denver et al., 2004a). A n atypical feature of the C. elegans genome is that (G/C)n of over 17nt are enriched relative to other eukaryotic genomes (Mike Schertzer, unpublished data). Furthermore, while (A/T)n follow the trend of decreasing abundance with increasing tract length, (G/C)n of over 8nt are observed at a much greater frequency than random expectation (Denver et al., 2004a). Longer (G/C)n are more prone to frameshift mutations (Denver et al., 2004a; Gragg et al., 2002), thus it is expected that they would be selected against. In addition, they require D O G 1 for their maintenance. Despite the energy expenditure associated with their maintenance, long (G/C)n occur throughout the genome in C. elegans, indicating that they may have special functions in this organism. In the C. elegans genome, the arms of the five autosomes are more densely populated by mononucleotide repeat tracts than the central core region (Denver et al., 2004a). This uneven distribution of is less pronounced on the X chromosome. Interestingly, the rate of recombination is also non-uniform along the autosomes, with the central core region (comprising about one third of each autosome) having reduced recombination rates relative to the arms (Barnes et al., 1995). The X chromosome, on the other hand, has more uniform rates of recombination along its length. This distribution could be a result of the meiotic crossover pattern. Or, a speculation arising from these observations is that mononucleotide repeats could have a role in meiotic recombination. Sen and Gilbert (1988) first showed that single-stranded G-rich D N A form parallel four-stranded structures in vitro and such association is believed to be mediated by guanine residues forming G-quartets, which are  73  planar structures where four guanine residues interact by Hoogsteen base-pairing. They proposed that during meiosis, homologous chromosomes recognize and associate with each other via a "zippering up" mechanism through joining of G-rich motifs along the chromosome among the four chromatids. A related proposal was put forth by Rosenbluth et al. (1990), who speculated that discrete genetic elements (alignment sites) along the chromosome are required for pairing of homologous chromosomes during meiosis. This proposal was based on the observation that in worm strains harboring certain deficiencies on the left half of chromosome V , recombination was inhibited on the side of the deficiency towards the center of the chromosome (Rosenbluth et al., 1990). (G)n would be a suitable candidate for facilitating homologous chromosome pairing via the mechanism proposed by Sen and Gilbert (1988) and Rosenbluth et al. (1990), because the two homologous chromosomes could align when the (G)n tracts on the four chromatids associate by forming G-quartets. (G)n are found along all of the chromosomes. Formation of G-quartet structures among the four chromatids might bring the homologous chromosomes in close proximity at sites where (G)n are present, and as a result, crossing over might occur preferentially at regions where (G/C)n are abundant, explaining the non-uniform and relatively uniform rates of recombination along autosomes and X chromosome, respectively. If (G/C)n do play a role in chromosome pairing during meiosis, their disruption might have an effect on recombination frequency. To test this idea, dog-l(gklO) has been tested for changes in recombination frequencies (Ann M . Rose, unpublished data). The prediction is that altered recombination frequencies will correlate with the occurrence of D N A breakage. In conclusion, DOG-1 is crucial for the maintenance of (G/C)n in the C. elegans genome: mutation in dog-1 results in genome-wide deletions specific to (G/C)n. Experimental data from studying dog-1 (gklO) (Cheung et al., 2002) and sequence comparison between DOG-1 and BACH1/BRIP1 (David L. Baillie, personal communication) suggest that DOG-1 is likely to be a helicase that unwind secondary structures formed on the lagging strand during D N A replication in C. elegans. Although DOG-1 is most similar to human BACH1/BRIP1 (David L. Baillie), it may not participate directly in the corresponding B R C A 1 D N A damage repair pathway that involves BRC-1 in C. elegans.  74  CHAPTER THREE  Single telomere length analysis (STELA) in C. elegans  3.1 Introduction Telomeres are in general composed of tandem G-rich repeats. In C. elegans, it has been shown that telomeric D N A consists of T T A G G C repeats spanning between 2 and 9 kb (Wicky et al., 1996; Lim et al., 2001; Benard et al., 2001; Ahmed et al., 2001). It has also been reported that telomeres of over lOkb are apparent in wild-type (Lim et al., 2001). The last chapter established that dog-1 is required for the maintenance of (G/C)n. In the absence of functional dog-1, mutants undergo sporadic deletions involving such tracts. Since telomeric D N A is in essence a long stretch of G-rich repeats, mutation in dog-1 may lead to similar deletion events in telomeric D N A , resulting in telomere shortening. This hypothesis was tested by measuring the telomere length in dog-1 (gklO) for a number of generations. The most commonly utilized technology for determining telomere length in various species is terminal restriction fragment (TRF) analysis. This approach involves a genomic Southern blot of terminal restriction fragments detected by hybridization to a probe containing telomeric repeats. While TRF analysis is of widespread utility, it has limited resolution and sensitivity (being capable of determining only the average telomere length of all chromosome ends from a large number of cells). Furthermore, telomere length measurement by TRF analysis can be confounded by the presence of interstitial telomeric sequences (ITS). Another common method for telomere length measurement is fluorescence in situ hybridization (FISH), which involves the hybridization of a fluorescent telomeric P N A probe to either metaphase spreads (Lansdorp et al., 1996) or interphase chromosomes of intact cells followed by flow cytometry (Rufer et al., 1998). In this approach, the intensity of fluorescent signals is proportional to telomere length. This method has the resolution and sensitivity to allow the analysis of all chromosome ends. Recently, a new PCR-based method called single telomere length analysis (STELA) has been developed that allows the measurement of single telomeres (Baird et al., 2003). Designed specifically for measuring the  A version of this chapter has been published. Cheung, I., Schertzer, M . , Baross, A . , Rose, A . M . , Lansdorp, P. M . , and Baird, D. M . (2004). Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans. Nucleic Acids Res 32, 3383-3391. 1  75  length of the X p Y p telomere in human cells, this method allows the accurate measurement of single telomere molecules thereby providing higher resolution than TRF analysis. Provided that telomere-adjacent D N A sequences are available, S T E L A is in principal applicable to telomeres of other human chromosomes and other species, allowing very accurate estimates of telomere lengths (Baird et al, 2003). To date the genome of C. elegans has been largely refractory to detailed telomere length analysis. This is primarily a consequence of the fact that the genome is interspersed with ITSs (C. elegans sequencing consortium, 1998). Hybridization of the telomeric probe to these sequences results in low molecular weight bands that interfere with detection and length measurement of short telomeres during TRF analysis (Wicky et al., 1996), and prevents the use of FISH-based methods in telomere length measurement in C. elegans (Elizabeth Chavez and Peter M . Lansdorp, unpublished data). Because S T E L A exploits the presence of unique sequences in the sub-telomeric region in amplification of telomeric D N A , it is not affected by the abundance of ITSs in the genome (Baird et al., 2003). This chapter describes the adaptation of S T E L A to C. elegans. With the sensitivity of STELA, telomere length can be measured from as few as a single worm.  3.2 Materials and Methods 3.2.1 Bulk genomic D N A isolation and ligation Worms were lysed for 1 hour at 55°C in lysis buffer A , which was made by combining 500ul N T E buffer (lOOmM NaCl, 50mM Tris, 20mM EDTA), 25ul of 10% SDS, lOul of lOmg/ml Proteinase K , and l u l p-mercaptoethanol. D N A was purified as follows: three extraction steps were performed with equal amounts of phenol/chloroform/isoamyl alcohol (25:24:1) in Phase Lock Gel™ tubes (Eppendorf). D N A was precipitated by addition of 68 pi 7.5 M ammonium acetate, 3 ul 20mg/ml glycogen and 500 pi 100% ethanol and incubation for 30 minutes on dry ice in ethanol, washed twice with 70% ethanol, and subsequently resuspended in lOmM TrisCl (pH8.5). Ligation was carried out as follows: a mix of 20ng of D N A (quantitated by PicoGreen® dsDNA Quantitation Kit, Molecular Probes) and l u l of l O u M telorette in a 2pi volume was incubated at 60°C for 10 minutes. Ligation was carried out at 35°C for 12-15 hours with 0.2ul of l U / u l T4 ligase (Amersham) and 0.4ul of 10X  76  manufacturer's ligation buffer in a final volume of 4pl. The ligase was then inactivated by incubation at 70°C for 15 minutes. 3.2.2 D N A isolation and ligation from 5 worms or single worms Five adult worms or a single adult worm were lyzed in 5 ul lysis buffer B ( I X P C R buffer (50mM Tris pH8.3, 35mM N a H P 0 , 0.5% Tween-20), 0.75 mfvl MgC12, 0.06 mg/ml 2  4  Proteinase K ) at 57°C for 60 minutes followed by incubation at 95°C for 15 minutes to inactive the enzyme. The lysate was diluted with lOmM TrisCl (pH 8.5) and D N A was extracted twice with equal amounts of phenol/chloroform/isoamyl alcohol (25:24:1) using Phase Lock Gel™ tubes (Eppendorf). D N A was precipitated by addition of 68 pi 7.5 M ammonium acetate, 3 pi 20mg/ml glycogen and 500 pi 100% ethanol and incubation overnight at -70°C, washed twice with 70% ethanol, and subsequently resuspended in 2ul water containing 2.5 u M telorette. The mixture of genomic D N A and telorette was incubated at 60°C for 10 minutes, followed by ligation at 35°C for 12-15 hours with 0.2ul of l U / p l T4 ligase (Amersham) and 0.3pi of 1 OX manufacturer's ligation buffer in a final volume of 3pi. The ligase was then inactivated by incubation at 70°C for 15 minutes. Genomic D N A was quantified by PicoGreen® dsDNA Quantification Kit (Molecular Probes). 3.2.3 Telomere amplification and detection Ligated D N A was used as template in original concentration or various dilutions in subsequent PCR reactions. A 15 pi PCR reaction contained the indicated amount of ligated D N A , I X PCR buffer IV (ABgene), 2mM M g C l , 0.5uM each of the teltail primer and the 2  subtelomeric region specific primer (512) (Table 1), 0.3mM of each dNTP (Amersham) and 1.5 U Extensor Hi-Fidelity P C R Enzyme Mix (ABgene). Thermal cycling conditions were the following: initial denaturation at 94°C for 3 min, 25 cycles of 94°C for 20 sec, 64°C for 30 sec, and 70°C for 8 min, followed by final elongation at 70°C for 10 min. P C R products were separated on 1% agarose gel, and transferred onto Hybond-N+ membrane (Amersham) according to standard alkali transfer protocol. Membranes were hybridized to either a [a32  P]dCTP random-labelled subtelomeric probe (fragment generated by PCR of N2 genomic  D N A with primers 509 and 510; see Figure 3-1) or a [y- P]dATP end-labelled ( G C C T A A ) 32  telomeric oligonucleotide probe in Church Buffer (0.25M pH7.2 sodium phosphate buffer,  77  5  l m M E D T A , 1%BSA, 7% SDS). Signals were detected using a Molecular Dynamics Storm 860 Phospholmager system (Amersham Biosciences). 3.2.4 Oligonucleotides The oligonucleotide sequences were as follows: teltail, 5' - T G C T C C G T G C A T C T G G C A T C - 3 ' ; 509, 5' - G T A G C A A G A A A G T G T C C T A G C G - 3 ' ; 510, 5' - G A C A G T A C T T A T G G G T T T C G T T - 3 ' ; 512, 5 ' - G A T G C G C A G C T A A C T A T A G G A - 3 ' ; telorette 502, 5' G A C A G C T A T G A C T G C T C C G T G C A T C T G G C A T C G C C T A A G - 3 ' ; telorette 503, 5 ' - G A C A G C T A T G A C T G C T C C G T G C A T C T G G C A T C T A A G C C T - 3 ' ; telorette 504, 5'-GAC A G C T A T G A C T G C T C C G T G C A T C T G G C A T C C C T A A G C - 3 ' ; telorette 505, 5 ' - G A C A G C T A T G A C T G C T C C G T G C A T C T G G C A T C C T A A G C C - 3 ' ; telorette 506, 5'-GACA G C T A T G A C T G C T C C G T G C A T C T G G C A T C A A G C C T A - 3 ' ; telorette 507, 5 ' - G A C A G C T A T G A C T G C T C C G T G C A T C T G G C A T C A G C C T A A - 3 ' .  3.3 Results and Discussion 3.3.1 Design of S T E L A Figure 3-1 shows the design of S T E L A in C. elegans. Chromosome V L was selected for two reasons. First, it has been shown that telomere length on different chromosome ends varies in C. elegans, and V L is among one of the shortest (Wicky et al., 1996). Second, unlike several other chromosome ends, the subtelomeric region of V L contains unique sequences for primer design (Wicky et al., 1996). Thus, single telomere analysis of this chromosome end should be both specific and well within the range of PCR. The method involves the ligation of an oligonucleotide, termed the telorette, to the 5' end of the telomere (i.e. the C-rich strand). The 3' end of the telorette, which is complementary to the G-rich overhang of the telomere, serves to facilitate ligation (Baird et al., 2003). The rest of the telorette has a unique sequence that is not found in the C. elegans genome to allow telorette-specific PCR amplifications. The terminus of the C-rich strand of the telomere may end in any of the six nucleotides within a telomeric repeat, which in C. elegans is T T A G G C (Wicky et al., 1996). Therefore, six telorettes were designed, each carrying one of the six possible  78  ChromdsomeVL  l e i b m e n e repeats - ^ i — I — S u b t e l o m e r i c r e g i o n t e l i a i t — .  .509 . A  telorette — f f i f t t S  la- I  I I  I  T- - I T-V  I  I  H  i  l  5 0 9 ' ^  509"-^  l  1 I I I  T-510 —  *  1.1 k b  Figure 3-1. Design of S T E L A in C. elegans. The diagram shows the telomeric repeats and subtelomeric region of chromosome V Left (VL). The 3' end of the telorette carries seven nucleotides that are complementary to the 3' G-rich overhang at the terminus of the telomere. Primer teltail aligns to the unique sequence of the telorette, and is used in amplification together with primer 512 that is specific to the subtelomeric region of chromosome V L . Primers 509 and 510 are used to generate a fragment for the subtelomeric probe. 509' and 509" are sequences with two and one mismatch from 509, respectively. The sequence of 509' is 5' G T A G C A A G A A A G T C T C C T A G G C C 3 ' on the + strand ; the sequence of 509" is 5 ' G T A G C A A G A A A G T A T C C T A G C C 3 ' on the + strand. (Figure adapted from Cheung et al, 2004)  frames of a telomeric repeat at its 3' end. Ligated genomic D N A is then amplified by PCR with the teltail and 512 primers. The primer teltail recognizes specifically the 3' end of the telorette (Baird et al., 2003), and the sequence of primer 512 is specific to the subtelomeric region of V L . Using these primers, the telomeric repeats and 1095 bp of subtelomeric sequence of V L are amplified. Such products would hybridize to a subtelomeric probe (generated by primers 509 and 510) as well as a telomeric probe that recognizes telomeric repeats (Figure 3-1). Preliminary analysis with all six telorettes demonstrated that telorette 503, whose 3' end has the frame of A A G C C T , provided the most efficient amplification, although the other five telorettes also supported amplification but to a lesser extend (data not shown). It is interesting to note that in humans, the telorette that provided the highest efficiency of telomere amplification (telorette2; Baird et al., 2003), ligated to the C-rich strand in the same frame as 503. It is thus possible that in both humans and C. elegans the C-rich strand preferentially terminates with the sequence 5'-AACCCT-3' and 5'-AAGCCT-3' respectively. The published sequence of cTel3X, which is the cosmid containing the telomere of V L ( C elegans sequencing consortium, 1998), predicts that PCR would generate a 300 bp product from primers 509 and 510. However, sequencing results revealed a duplication of 170 bp within the 509/510 fragment in the reference wild-type strain N2 and a number of other strains originating from different laboratories. Therefore, the true sequence of cTel3X in N2 is likely to be as shown in Figure 3-2 (GenBank accession AY559143).  3.3.2 S T E L A from bulk genomic D N A Figure 3-3 shows the sensitivity of S T E L A in measuring telomere length in C. elegans. When ~300pg of bulk genomic D N A from the wild-type strain N2 was used in a reaction, a smear was observed. With decreasing amount of template D N A , the smear was resolved into discrete bands, each band representing a single amplified telomere. As each telomeric molecule contains 1.1 kb of telomere-adjacent D N A , bands below this size should not occur. When D N A was ligated at 5 ng/uL, as little as lOpg was needed for PCR amplification, thus D N A extracted from a single plate (typically containing 200-300 worms) is more than sufficient for telomere length measurement. There are several features shown in  80  A 5' gtagcaagaaagtgtcctagc^taaAaataggaaatttttcgctctttcagaaCAGTATACT^TGTCTCTGTACCG gtctctgtaccgacgatattc^tttcaaaaatcgcaaaaaaagtttttttcaaaatacc^ aattatttttgattgtttatatttaagtagcaagaaagtgtcctagcctaaaaataggaaattttttgctctttcagatc agtatactaaacattc^cgtcrtggattaaaattaaaaaggtagtgttaaataattaaaaatcatttagggggga^ cccataagtactgtc3' B Telomere  (previously published sequence)  3'  Telomere  {sequence found in this study)  Figure 3-2. The fragment amplified using the 509 and 510 primers (509/510) contains a duplication that is not present in the previously published sequence of cTe!3X. (A) Sequence of the 509/510 fragment. The previously published sequence is shown in lowercase, and nucleotides not present in the previously published sequence are in uppercase letters. (B) A schematic representation of the duplication. The grey box represents the duplicated sequence. The two duplicated sequences are separated by a C, and are almost identical except for an insertion of T. (Figure adapted from Cheung et al., 2004)  Figure 3-3 that demonstrate the specificity of S T E L A in amplifying telomeric sequences. First, almost every band could be detected by hybridization with both the telomeric and subtelomeric probes. Occasional bands were observed that showed differential hybridisation with the two probes being only detected by the subtelomeric probe. Such bands were below the threshold length (~1.2kb) that facilitated detection by the telomere probe, presumably as a consequence of a suitable telomere target sequence. Secondly, the number of bands amplified per reaction was proportional to the amount of D N A template. Thirdly, bands had consistent intensities suggesting that all molecules were amplified from the first round of PCR. These data are consistent with specific amplification from single telomeric molecules. 3.3.3 S T E L A from 5 worms or single worms One goal in developing S T E L A for C. elegans was to measure telomere length from a few or even single worms. TRF analysis in C. elegans requires on average 5 pg of genomic D N A for each measurement, thus thousands of worms have to be grown for D N A extraction. 81  Mutants with telomere defects are likely to have progressively reducing brood size (Ahmed and Hodgkin, 2000; Herrera et al., 1999; Lee et a l , 1998), and collecting thousands of such mutants for studying telomere length dynamics may sometimes be impractical. For C. elegans to become a valuable model organism in studying telomere biology, a technique with higher sensitivity than TRF analysis is required. To explore the possibility of performing S T E L A starting from a small number of worms, D N A was extracted from five worm samples and subjected to S T E L A (Figure 3-4). The C. elegans genome is -100 million base pairs, and an adult hermaphrodite has 959 somatic cells and -1000 germ nuclei from each of the two  lOOpg 3kb-  -1.9kb  *  •  l.6kb  B  -fl9kb  -O.bkb  3kb-  1.9kb  2kb  -0.9kb  I ,f>kn-  0.5 kb  ikb  Figure 3-3. S T E L A from bulk N2 genomic DNA. 300pg, lOOpg, 30pg, lOpg, or 3pg of N2 genomic D N A ligated to telorette 503 was amplified with primers teltail and 512. Five identical reactions were set up for each amount of D N A template. Size is indicated on the left, and the corresponding telomere length is indicated on the right. The blot was hybridized to (A) the subtelomeric probe, or (B) the telomeric probe. (Adapted from Cheung et al., 2004)  82  16kb-  -0.5tcb  Figure 3-4. Telomere length measurement from 5 worms by STELA. D N A was extracted from 5-worm samples independently. After ligation to the telorette, D N A was pooled, and D N A from an equivalent of 0.8 worm, 0.25 worm, or 0.08 worm was amplified. Assuming each adult worm contains ~400pg of genomic D N A , they would represent ~320pg, ~100pg and ~30pg D N A respectively i f D N A extraction was 100% efficient. Five identical reactions were set up for each amount of D N A template used. The marker lane is shown on the left and the corresponding telomere length is shown on the right. The blot was hybridized to (A) the subtelomeric probe, or (B) the telomeric probe. (Figure adapted from Cheung et al., 2004)  gonadal arms (Berry et al., 1997; Sulston, 1988). Therefore, each worm was estimated to contain approximately 400pg of D N A . Quantification of genomic D N A extracted from 5worm samples showed that on average 1700pg was obtained (data not shown), demonstrating  83  that extract was reasonably efficient. Different worm equivalents of D N A ligated to the telorette were used in PCR reactions. A smear that resembled a TRF analysis (Ahmed and Hodgkin, 2000; Wicky et al., 1996) was generated when D N A from an equivalent of 0.8 worm was used in PCR amplification, and resolved into a single banding pattern upon serial dilution to 0.08 worms per PCR (Figure 3-4). Figure 3-5 shows that S T E L A was sensitive enough to allow telomere length measurement from as little as a single adult worm.  0.2worm  1.6kb-  I  Eg,  02wocm  D  -O.Skb  £  3kb-  -l.9kb  .  :).<>kh  L6kb-  ^kb  §  -0.5kb  Ikb-  Figure 3-5. A single worm is sufficient for telomere length measurement by STELA. D N A was extracted from a single adult worm. After ligation to the telorette, D N A from an equivalent of 0.2 worm was amplified. Assuming each adult worm contains ~400pg genomic D N A , it would represent 80pg of D N A if D N A extraction was 100% efficient. Four identical reactions were set up. The marker lane is shown on the left and the corresponding telomere length is shown on the right. The blot was hybridized to (A) the subtelomeric probe, or (B) the telomeric probe. (Figure adapted from Cheung et al., 2004)  84  3.3.4 Use of S T E L A to measure telomere shortening in mutants As a further validation of the technique, a mutant with known telomere defects was subjected to S T E L A . The C. elegans mrt-2 gene is a homolog of the S. cerevisiae RAD17 and S. pombe radl+ checkpoint genes (Ahmed and Hodgkin, 2000). Mrt-2 shows progressively shortening telomeres and frequent end-to-end chromosome fusions (Ahmed and Hodgkin, 2000). S T E L A revealed that V L telomere shortened by approximately 0.7 kb within seven generations in mrt-2 (Table 3-1, Figure 3-6). This was comparable to that determined by TRF analysis (Ahmed and Hodgkin, 2000), which measures average telomere length among all telomere ends. This result demonstrates that the dynamics of V L telomere length could represent the average of all telomeres in certain strains. A n interesting observation from our analysis was that the distribution of telomere lengths in mrt-2 in any given generation was less heterogeneous than those of the wild-type strains (Table 3-1, Figure 3-6). Furthermore, SD was very similar among the three generations examined in spite of the changes in telomere length (Table 3-1). Therefore, the mrt-2 gene may contribute to telomere length heterogeneity in C. elegans.  85  12  F9  FS  -2.9kb  3kb- IMP  2kb-  1.9kb  l,6kb-  ikb-  B  0.9xt)  HP  m  -05kb  3kb  -2.9kb  2kb-  1.9kb  1.6kb-  0.9kb  Ikb-  0.5  1.0  1.5  telomere length (kb)  86  20  Figure 3-6. Telomere shortening in mrt-2. (A,B) Telomere length in five worms from F2, F5, and F9 was analyzed by STELA. Each lane was amplified from an equivalent of 0.1 worm. For each generation, seven identical reactions were set up. The marker lane is shown on the left and the corresponding telomere length is shown on the right. The blot was hybridized to (A) the subtelomeric probe, or (B) the telomeric probe. (C) Distribution of telomere length at each generation in mrt-2. Intensities of signals of each lane in (A) was measured, and those from the same generation were added together at each telomere length interval. Relative intensity was plotted against telomere length. The thick line represents F2, the thin line represents F5, and the dotted line represents F9. Relative intensity may represent frequency in this case because the blots were hybridized to the subtelomeric probe and variations in band intensities should be random among all bands. (Figure adapted from Cheung et al., 2004)  Table 3-1. Distribution of telomere length in N2 and mrt-2 (Table adapted from Cheung et al., 2004) Strain N2  a  V L Mean telomere length (kb) 1.25 (±0.05)  b  S.D. 0.34  b  Mrt-2 F2  1.19 (±0.07)  c  0.11  c  Mrt-2 F5  0.86 (±0.05)  0  0.10  c  Mrt-2 F9  0.46 (±0.04)  0  0.08  c  a) The original N2 strain b) Calculated from S T E L A of different generations of N2 (Figure 4-1) c) Calculated from Figure 3-6 (A)  87  C H A P T E R FOUR  Use of S T E L A in studying telomere biology in C. elegans  4.1 Introduction C. elegans telomeres were first characterized by Wicky et al. (1996), who found that they are composed of T T A G G C repeats spanning 4 to 9kb. However, the length of telomeres as analyzed by TRF analysis in N2 is inconsistent among different laboratories: Ahmed and Hodgkin (2000) reported that N2 telomeres span between 2 and 7 kb, while shorter telomeres of between 2 and 4 kb were found by others (Benard et al., 2001). At the other extreme, Lim et al. (2001) observed telomeres of over lOkb in N2. These results suggest that environmental, genetic and epigenetic factors may play an important role in telomere length homeostasis in C. elegans. To date only two genes have been documented to be required for telomere maintenance in C. elegans: mrt-2 and clk-2. mrt-2 is the homologue of the S. cerevisiae RAD17 and S. pombe radl checkpoint genes, mrt-2 mutants are hypersensitive to X-rays and display progressive telomere shortening (Ahmed and Hodgkin, 2000). clk-2 is the C. elegans homologue of yeast tel2 (Benard et al., 2001; Lim et al., 2001), mutation in which has been shown to result in short but stable telomere length (Runge and Zakian, 1996). There have been conflicting results concerning the role of clk-2 in telomere maintenance (Ahmed et al., 2001; Benard et al., 2001; Lim et al., 2001), which may be related to the observation that telomere length tends to fluctuate in C. elegans (Ahmed et al., 2001; this chapter). The previous chapter described the development of S T E L A for C. elegans prompted by the need for a more sensitive technique for telomere length measurement in this model organism. This chapter describes the characterization of wild-type chromosome V left (VL) telomeres by S T E L A to illustrate the unique opportunities in studying telomeres in C. elegans made available by the technique. The telomerase reverse transcriptase mutant trt-1 will be characterized. Lastly, telomere length will be examined in dog-1 (gklO) to see i f dog-1 is also required for maintaining telomeric D N A .  A portion of this chapter has been published. Cheung, I., Schertzer, M . , Baross, A . , Rose, A . M . , Lansdorp, P. M . , and Baird, D. M . (2004). Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans. Nucleic Acids Res 32, 3383-3391. 1  88  4.2 Materials and Methods 4.2.1 Strains Worms were handled as described (Brenner, 1974) but were grown at room temperature (19 °C to 23°C) unless stated otherwise. The strains used in this chapter were the following: N2, glp-4(bn2ts) (KR4138 and KR4139), trt-l(ok410) (KR4050), and different isolates of wild-type C. elegans obtained from the Caenorhabditis Genetics Center: CB3191, CB3192, CB4852, CB4856, CC2, TR403, N2, and N2(ancestral). 4.2.2 Genotyping of trt-1 To check the genotype of trt-1, three primers were used: 487, 488, and 489. The sequences of these primers were: 497, 5 ' - G T G C T C A T T T A A G T C T C G T C A G A - 3 ' ; 488, 5'T T C C T C T T C A C A T T T G G A T C C A C - 3 ' ; 489, 5' - G A G T T A C G A C A A T C T G A C C T T G A - 3 ' . 487 and 489 flank the 1443bp deletion in ok410. 488 recognized a sequence located within the deletion and could generate a product with 487. Hence, two products could potentially be amplified in W T by the three primers: a 2403bp product generated by 487 and 489, and a 652bp product generated by 488 and 489. The 652bp product was amplified preferentially and the 2403bp product could not be detected. Because of the absence of the recognition site for 488 and an insertion of 13bp in ok410, only one product of 973bp could be produced. After worm lysis, the lysate was used directly as template in PCR as described in section 2.2.3 except that primers 487,488 and 489 were used. Thermal cycling conditions were the following: initial denaturation at 95°C for 4 min, 34 cycles of 95°C for 30 sec, 62°C for 30 sec, and 72°C for 1 min, followed by final elongation at 72°C for 10 min. PCR products were separated on 1% agarose gel and D N A was visualized by ethidium bromide. 4.2.3 S T E L A S T E L A for telomere length measurement of chromosome V L was carried out as described in Chapter 3.  89  4.2.4 Analysis of S T E L A data From a gel file generated by the Phosphorlmager, the intensity of signals was measured (in 0.1mm intervals) along a lane by ImageQuant 5.0 software. Data was imported into Microsoft Excel for size analysis. From a size marker lane, a plot of size (kb) against distance (mm) was generated and fitted by the power function (y=ax ), which works best for bands b  <5kb. The formula was then used to calculate the size, and 1.095kb was subtracted to obtain telomere length for each measured intensity value. A plot of signal intensity or relative intensity (%) was plotted against telomere length (kb) by Prism 3.0. Relative intensity was calculated by dividing intensity by the highest value and multiplied by 100%. To calculate the rate of V L telomere shortening in trt-1 mutants (section 4.3.5), plots of intensity against telomere length were generated for each generation in each line from Figure 5. In most cases where a major cluster of bands was amplified, telomere length for the generation was determined to be the peak of the plot. In cases where bands were spread out, telomere length was arbitrarily determined to be the middle of the spread. Telomere length determined this way was plotted against generation and the plot was fitted by linear regression. The rate of V L telomere shortening is the slope of the linear curve.  4.3 Results 4.3.1 Fluctuations of telomere length in N2 Telomere length must be maintained for the survival of the species. Fluctuations in telomere length and a tendency for telomeres to elongate over the generations have been observed by Ahmed et al. (2001). To see i f these are general phenomena occurring in wild-type C. elegans, N2 worms from F I , F5, F9, F13 and F17 were analyzed by S T E L A . For each generation, 5 worms were sampled as an overall representation of the particular generation. This experiment may also serve as a reference when studying telomere phenotypes in different strains. Figure 4-1 shows that indeed there are slight fluctuations. Despite these fluctuations, V L telomeres were maintained within a range of ~lkb (~1 to 2kb) in all generations examined, indicating that telomere length was under relatively tight control that was chromosome-specific.  90  4.3.2 Telomere length heterogeneity within clonal population Results from Chapter 3 and above show that the majority of V L telomeres in N2 span a range of approximately 1 kb. This range reflects the sum of telomere length heterogeneity in all individual worms being analyzed. To examine variations in the distribution of telomere length among individuals, a hermaphrodite parent and 10 of its progeny were analyzed separately by S T E L A (Figure 4-2). While most of the progeny exhibited a similar distribution of telomere length as the parent, there are individuals (progeny 3 and 9, Figure 42) with obvious V L telomere length elongation. On the other hand, in one of the progeny examined (progeny 6, Figure 4-2), the majority of V L telomeres became shorter although the range of telomere sizes was still comparable to the parent. Hence, considerable telomere length variation can occur within one generation.  Figure 4-1. Telomere length fluctuations in wild-type C. elegans. Telomere length of V L was measured by S T E L A at F l , F5, F9, F13 and F17 (method as described in Chapter 3). D N A was extracted from 5 reproductive stage adult sampled at each of the generations. D N A was ligated to the telorette and 0.1 worm equivalent was used in each PCR reaction. Five identical reactions were set up for each generation. Marker lane is shown on the left and the corresponding telomere length is indicated on the right. The blot was hybridized to the subtelomeric probe.  91  4.3.3 Evidence of tissue-specific telomere length Higher eukaryotes, including mouse and human, exhibit tissue-specific telomere lengths, which may be caused in part by different levels of telomerase activities (CovielloMcLaughlin and Prowse, 1997; Greider, 1998; Hastie et al., 1990). In humans, reproductive tissues in the testes have the highest telomerase expression and longer telomere length than most somatic tissues (Bekaert et al., 2004; Kolquist et al., 1998). To see i f telomere lengths in germline and somatic tissues are also different in C. elegans, the temperature-sensitive mutant glp-4(bn2ts) was employed. In glp-4(bn2ts), germ cells fail to enter meiosis and the mutant becomes essentially germline-less when grown at the restrictive temperature of 25°C (Beanan and Strome, 1992). glp-4(bn2ts) parents were allowed to lay eggs separately first at the permissive temperature of 14°C and then at 25°C, and telomere length was analyzed and  l,6kb-  *0.5kb  Figure 4-2. Telomere length heterogeneity in wild-type C. elegans. A n N2 parent and 10 of its progeny were analyzed by S T E L A (method as described in Chapter 3). D N A was extracted from each single worm, ligated to telorette, and the entire D N A sample was used as template in PCR. Therefore, each lane represents a single worm. The blot was hybridized to the subtelomeric probe.  92  C 2Sdeg germline e s i  14deg germline  <ktv  J f, fi fi ±  25deg germline leu  1  1  14deg germline  5kb5kb- ~  -Z 2.9kb  fi  -3.9kb  •  4kb- * 3kb-  1.9kb 3kb-  M. ' 1  3kl>  2kb-  0.9M>  1.6kb-  -03kb  B germlin*-(eM  germiin*  2.9kb  3kb-  I.Qfcb  1 6kb-  1.6kb-  -t: <i*.r.  " ' 1  •  •  -O.Skb  -•-.d,-i qfrn iae  ikb  2.9kb  3kb  2kb  -1.9kb  *  1 6kb  cskn  9 k b  0.9kb  2Sdeq germline-lew  * _JJ  4kb  2kb-  2kb-  D  It  2 9kb  JL  -•  "  J% *  -0.9kb -0,5kb  Figure 4-3. Telomere length pattern in the presence or absence of germline. glp-4(bn2ts) parents were allowed to lay eggs first at 14°C and then at 25°C. Progeny grown at 14°C developed a germline, while progeny grown at °C were germline-less. Eight adult progeny was analyzed by S T E L A (method as described in Chapter 3). Panels A and B are different generations of the same line of glp-4(bn2ts); panels C and D are different generation of another line of glp-4(bn2ts). Each lane represents a single worm. The blots were hybridized to the subtelomeric probe.  compared among adult siblings grown at the two different temperatures. Results from four parents are shown in Figure 4-3. Comparison between glp-4(bn2ts) progeny growing at 14°C and 25°C by visual examination of the blots reveals that in 3 out of the 4 experiments (panels A , C and D in Figure 4-3), discrete telomere bands that are longer than the majority were  93  relatively abundant in progeny growing at 14°C, but were absent in most individuals growing at 25°C. These telomeres most likely came from the germline, suggesting the presence of mechanisms in elongating telomeres in the germline. The majority of the telomeres fell within the same range for progeny grown at the two different temperatures. 4.3.4 Variations in telomere length in wild-type C. elegans strains Telomere length of V L in the N2 strain cultured in our laboratory had a mean of 1.25±0.05kb and a SD of 0.34 (Table 3-1). To determine how typical the V L telomere length was of wildtype C. elegans, S T E L A was applied to a number of different wild-type strains, including the reference wild-type strain N2, obtained from the Caenorhabditis Genetics Center (CGC, Minneapolis, M N ; a description of the origin of some of the wild-type strains can be found in Hodgkin and Doniach (1997). D N A was extracted from 5 worms from each strain and ligated to the telorette and an equivalent of 0.8 worm, 0.25 worm, or 0.08 worm was subjected to S T E L A . Figure 4-4 shows the variations of V L telomere length in eight wild-type strains. Detailed telomere length analysis revealed that N2 had the shortest mean telomere length (1.49±0.13kb) among the eight strains (Table 4-1). CB3192, CB4852, CB4856, and N2(ancestral) had similar telomere length distribution and slightly longer mean telomere length than N2. In CB3191 and CC2, telomere length was more than twice as long as N2. The subtelomeric region was likely to be invariant among these different wild-type strains because the length of the subtelomeric fragment amplified by primers 509 and 512 was the same as in N2 (data not shown). A n exception was TR403, where the 509/512 fragment was about 340bp shorter than N2 (data not shown). Sequencing results suggests that a shorter fragment was amplified because an alternative recognition site for primer 509 was used (Figure 3-1), indicating that a mutation at the normal primer 509 recognition site might have occurred, leading to the use of a site with one mismatch. Interestingly, amplification of V L telomere in TR403 resulted in bands that were extremely large (>10 kb). These large bands could be resulted from either extremely long telomeres of over 10 kb in TR403, or a long insertion between the site of primer 509 and the telomeric repeats. The later possibility was ruled out because the telomeric probe could hybridize to bands that were over 2 kb in size, indicating the subtelomeric fragment amplified must be less than 2 kb. Such extremely long telomeres in TR403 relative to other wild-type strains suggest that intra-species genetic variations could dramatically affect telomere lengths in C. elegans.  94  CB3191  •I  1 2 3 4 5 6 7 8910  %&m • 5W> 1 4kb7 k b  2kbl,6kb-  CB3192  CB4852  1 2 3 4 5 6 78910  1 2 3 4 5 6 7 8910  CB4856  1 2 3 4 5 6 7 89 10 |  I  -5.9kb -3 9kb  * 1  •  *  S i  *  -09kb -O.Skfl  -3.9kb  5 k b  -0.9kb 1 A k b  -  -0.5kb  CC2  TR403  N2  1 2 3 4 5 6 78910  1 2 3 4 5 6 7 8910  N2 (ancestral)  § b u  S  1 2 3 4 S6 7 8910  1 2 3 4 5 6 7 89 10 | J  I  -5.9kb 3.9kb -29kb 1.9kb  -  0.9kb  *  -O.SM)  7kb 5kb4kb3kb  5.9kb -3.9kb 2.9kb II S I  2kb16kb  -• J  i f t &• f#c•f t • £ -0-9kb  »)5  CB3191  0  2  4  CB3192  6  8  2  Telomere length (kb)  4  6  8  Telomere length (kb)  CB4852  CB4856  3? toon 9  w  telomere length (kb)  Telomere length (kb)  CC2  TR403  0 Telomere length (kb)  2  4  6  Telomere length (kb)  N2  N2(ancestral)  100i  0  2  4  6  8  Telomere length (kb)  Telomere length (kb)  Figure 4-4. Variations in the length of V L telomeres in different wild-type C. elegans strains. The wild-type strains (A,B) CB3191, CB3192, CB4852, CB4856, and (C,D) CC2, TR403, N2, N2(ancestral) were analyzed by STELA. D N A was extracted from 5 worms for  96  each strain. After ligation to the telorette, D N A from an equivalent of 0.8 worm (lanes 1,2), 0.25 worm (lanes 3-6) or 0.08 worm (lanes 7-10) was amplified. In TR403, high molecular weight bands could not be amplified in lanes 1 and 2 as in all other lanes. The marker lane is shown on the left and the corresponding telomere length is shown on the right. The blot was hybridized to (A,C) the subtelomeric probe, or (B,D) the telomeric probe. (E) Distribution of telomere length in different wild-type strains. Intensities of signals from lanes 3 to 10 of each blot from (A) and (C) were measured and added together at each telomere length interval. Relative intensity (compared to the highest intensity) was plotted against telomere length. Relative intensity may represent frequency in this case because the blots were hybridized to the subtelomeric probe and variations in band intensities should be random among all bands. (Figure adapted from Cheung et al., 2004)  Table 4-1. Distribution of telomere length in different wild-type C. elegans strains (Table adapated from Cheung et al., 2004)  Strain  a  V L Mean telomere length (kb)  S.D.  N2  b  1.25 (±0.05)  d  0.34  d  N2  C  1.49 (±0.13)  e  0.49  e  N2 (ancestral)  1.80 (±0.14)  e  CB3192  1.88 (±0.19)  e  0.56  e  CB4852  2.00 (±0.26)  e  0.73  e  CB4856  2.05 (±0.17)  6  0.48  0.48  e  6  a) CB3191, CC2 and TR403 were not analyzed b) The original N2 strain c) Fresh N2 strain from the Caenorhabditis Genetics Center (obtained in Nov, 2003) d) Calculated from S T E L A of different generations of N2 (Figure 4-1) e) Calculated from lanes 3 to 10 of the corresponding blots in Fig 4A,C  97  4.3.5 Progressive telomere shortening in trt-l(ok410) The putative reverse transcriptase component of C. elegans telomerase is encoded by trt-1 (DY3.4) of Chromosome I (Malik et al., 2000). The allele ok410 is a 1443bp deletion with a 13bp insertion between the breakpoints (generated by the C. elegans gene knockout facility at Oklahoma Medical Research Foundation). This deletion spans the region that encodes for 3 of the 6 conserved reverse transcriptase motifs as predicted by Malik et al. (2000). It is expected that the mutant protein would completely lack of catalytic activity because of the extensive deletion. 16 lines of trt-1(ok410) were passaged until the line became sterile. Starting from F2, the number of adult progeny was counted for each generation, and the parent was analyzed by S T E L A after all eggs were laid. Progeny size was reduced significantly relative to wildtype but varied widely among the 16 lines (Table 4-2). Despite the reduced progeny size, most worms appeared healthy in early generations. In late generations, worms appeared sluggish and early death was frequent (data not shown). There was transient increase in progeny size in many lines (#3-7, 13). A possible explanation for this observation is that L4 animals were transferred in the experiments. In doing so, unhealthy, slow-growing individuals producing smaller-than-average brood sizes were probably selected against. This bias could be minimized by randomly transferring multiple worms at an earlier larval stage; however, such practice would reduce the resolution of S T E L A analyses because of the higher variation in expected telomere length. Consistent with TRT-1 being a component of telomerase, all 16 lines of trt-1(ok410) displayed progressive shortening of telomeres (Figure 4-5).The rate of telomere shortening in each line was calculated by arbitrarily determining the mean telomere length for each generation, which was then analyzed by linear regression (Table 4-3). In most lines, the V L telomere shortened between 100 and 150bp per generation with a range of 70bp to 260bp per generation. The frequency of different amounts of telomeric D N A lost between generations was also determined. Figure 4-6 shows that consistent with the rate of telomere shortening, a loss of 1 lObp to 150bp of telomeric D N A per generation was the most frequent. In a number of instances, V L telomere did not appear to shorten from one generation to the next (for example, F3 to F4 in line 6 and F2 to F3 in line 11; Figure 4-5). It is possible that in those  98  66  Figure 4-5. Progressive telomere shortening in trt-l(ok410). Trt-l(ok410) was outcrossed to N2 males. From 2 heterozygous parents, 16 homozygous trt-1 lines were set up separately. For each generation, the parent (post-reproductive stage) was analyzed by S T E L A (method as described in Chapter 3), starting from F2 and ending at the generation that became sterile. Generation numbers are indicated on top of each panel; the heterozygous parent is considered as Po. A number was assigned for each line (1 to 16) and it is shown at the top right corner of each panel. A l l blots were hybridized to the subtelomeric probes  cases, rare long telomeres (elongated by telomerase-independent mechanism) were inherited, resulting in no apparent telomere shortening between those generations. 4.3.6 Reduced telomere length heterogeneity in trt-1 (ok410) Telomere length heterogeneity in trt-1 was studied by S T E L A on individual progeny from a single parent (Figure 4-7). In contrast to wild-type, telomere length was highly uniform among trt-1 (ok410) progeny. N2 progeny had both elongated and shortened telomeres relative to the parent (Figure 4-2). The two clusters of bands in most worms shown in Figure 4-7 (e.g. progeny 1, 3, 5, 7, and 8 in Figure 4-7A) likely represent the two alleles of V L . In order to maintain the strain, trt-1 (ok410) has to be routinely outcrossed. Thus, a longer V L telomere from the wild-type parent might be maintained at a longer length separately from the shorter V L telomere from the trt-1 parent. Segregation of the two telomeres with distinctive lengths would result in progeny with two long telomeres (eg progeny 2 of Figure 4-7(A)), two short telomeres (progeny 6 of Fig 4-7(A)), or one long and one short telomere (progeny 1, 3, 5, 7, 8 of Fig 4-7(A)). In addition, trt-1 (ok410) lacks the abundance of long telomeres seen in wild-type. The lack of long telomeres in trt-1 (ok410) is evidence for their elongation by telomerase in wildtype. The abundance of the longer telomeres in wild-type sometimes results in a long smear of bands (data not shown). In trt-l(ok410), occasional telomeres up to ~ l k b longer than the clusters could be observed, indicating that telomerase-independent telomere-lengthening mechanisms are present in C. elegans. Another characteristic of trt-1 (ok410) is the high frequency of short outlying telomeres relative to wild-type. Although it was not present in every individual, there was a significant number of trt-1(ok410) worms in which a ladder of short outlying bands were amplified. These short outliers were more apparent in mutants with longer telomeres (compare panels A and B with C and D in Figure 4-7).  100  Table 4-2. Progeny size in 16 lines of trt-1 (ok410)  Generation  Number of adult progeny  1  2 *3 4 5  98 96 62 27  2  2  168  3  2 *3 4 5  58 14 58 0  2 3 4 5 6  154 101 16 135 84  2 3 *4 5  135 209 158 16  Line  4  5  6  a  2 3 4 5 *6 7 8  130 153 180 10 84 8 1  Generation  Number of adult progeny  7  2 3 4  57 174 101  8  *2 3 4  49 34 32  9  2 3 4 5 6  177 179 97 18 26  10  2 3  176 27  11  2  20  12  2 3  173 13  13  2 3 4  182 23 81  14  2 3  230 204  15  2  183  16  *2 3  120 19  Line  a  a. Line number is according to Figure 4-5 * Possible underestimation due to various reasons, such as early death of parent  101  Table 4-3. Rate of V L telomere shortening (kb/generation) in trt-l(ok410)  Line 1 2 3 4 5 6 7 8i 8ii 9 10 11 12 13 14 15 16 c  c  a  Rate of V L telomere shortening (kb/generation) b  0.17 0.26 0.14 0.12 0.09 0.09 0.07 0.11 0.14 0.12 0.12 0.1 0.15 0.15 ND 0.2 0.12  a. line number according to Figure 4-5 b. signals from Figure 4-5 were measured using ImageQuant software and plotted as a function of telomere length. In most cases where a major cluster of bands weas amplified, telomere length for the generation was determined to be the peak of the plot. In cases where bands were spread out, telomere length was arbitrarily determined to be the middle of the spread. c. i and ii refer to the two distinct clusters of bands in line 8. See Figure 4-5. d. assumption in calculating the rate was that the top cluster of bands in F3 arose from some rare longer telomeres that were not amplified from F2. Therefore, the top cluster in F3 was excluded from the analysis. N.D. not determined  102  25  0.00-0.05  0.06-0.10  0.11-0.15  0.16-0.20  0.21-0.25  0.26-0.30  Telomeric DNA loss (kb)  Figure 4-6. Frequency of different amounts of telomeric D N A lost between generations. From Figure 4-5, mean telomere length was determined for each generation in each trtl(ok410) line (except line 14). The difference in mean telomere length between subsequent generations was calculated and sorted into the indicated categories.  4.3.7 No apparent telomere phenotype in dog-l(gklO) Chapter 2 described that DOG-1, a putative DExH-box helicase, is required for maintenance of (G/C)n sequenes. It has been proposed that DOG-1 resolves secondary structures formed by G-rich single-stranded D N A during lagging strand synthesis (Cheung et al., 2002). Since telomeric repeats are G-rich, it is possible that telomeric D N A would also form secondary structures during lagging strand synthesis. In such case, dog-1(gklO) would undergo sporadic deletions at telomeres, resulting in telomere shortening and a higher frequency of short outlying telomeres. Terminal restriction fragment analysis showed that telomeres seemed to shorten progressively in dog-1 (gklO) (supplementary information in Cheung et la (2002); Appendix E). However, limitations of TRF analysis (relatively low sensitivity and interference from ITSs) prevent a definitive conclusion as to whether dog-1 (gklO) displays any telomere defects. Therefore, S T E L A was used to examine telomere lengths in dogl(gkl0). dog-1 (gklO) was passaged for a number of generations and 5 worms were sampled from each generation. Figure 4-8 shows S T E L A results of two separate lines for dogl(gkl0). Neither telomere shortening nor a higher frequency of short outlying bands could be observed. In fact, there seemed to be slight telomere lengthening over the generations. For the dog-1 (gklO) line represented by Figure 4-8B, it is evident that some elongated telomeres  103  arose at around F l 1, when telomeres of lengths comparable to earlier generations were still present. These elongated telomeres were inherited and maintained, while the shorter telomeres were lost in later generations (probably by chance), resulting in the distribution of telomere lengths seen in F14. Given that telomere length fluctuates in wild-type (Figure 4-1),  L  E = o c  QI  S . 5kb-  1 2 3 4 5 6 7 8  4kb-  2kb-  f  •1  * ¥ *  a  • *  , „ , , -3.9kb  c  ,. 1 2 3 4 5 6 / 8 5kb- •  -3.9kb  -2.9kb  4kb m  -2.9kb  -1.9kb  3kb- < H ^ ^ ^ ^ ^ H f c - i . 9 k b  2  I  1 6 f c b  6 7 8 * " -3.9kb -Z9kb  3kb  1.9kb  a Skb .  4 k b  3kh  2kb1.6kb-  b  -  "O^b -O-Skb  D  I ? 1 2 3 4 5  k  I  -0.5kb  .8  £ JZ r »• o v  CT o  -0.9kb  1.6kb-  •jklj 4kb  B  If  1 2 3 4 5 6 7 8 9 10 111213141516 _ -3.9kb 2 i 9 k b  -19kb  0.9kb MPw  -0.5kb  16k  [3  05kb  Figure 4-7. Reduced telomere length heterogeneity in trt-1 (ok410). 8 or 16 reproductivestage progeny from 4 separate parents (A to D ) were analyzed by S T E L A (method as described in Chapter 3). Each lane represents a single progeny. A l l blots were hybridized to the subtelomeric probe.  104  and it has been documented that telomere lengths in C. elegans tend to increase over the generations (Ahmed and Hodgkin, 2000), the slight telomere lengthening in dog-1 (gklO) was not deemed significant and a telomere defect could not be assigned to the mutant strain.  o  F4  j  F6  Skb * 4kb- *  2kb- *  F8  9  F10 3.9kb -2.9kb  m  *  -0.9kb  16kb  0.5kb  u.  B  FS  Ml  F H  \ 14  Skb- <•> 4kb- «•>  3kb  | | ' 3.9kb  « M  _ «»,  •  -29kb  * v •  w  . -l.9kb  2kb  -09kb  1.6kb-  -0-5kb  Figure 4-8. No apparent telomere defects in dog-1 (gklO). 5 worms from each of the indicated generations were sampled. D N A was extracted and ligated to telorette. 0.1 worm equivalent of D N A was used in each PCR reaction (method as described in Chapter 3). Five identical reactions were set up for each generation. Two separate lines (A and B) are shown here. Blots were hybridized to the subtelomeric probe.  105  4.4 Discussion 4.4.1 Telomere length variations in C. elegans Results from S T E L A illustrated that telomere length was highly variable (varying between 1.25 kb to >10 kb) among different wild-type C. elegans strains (Cheung et al., 2004). This variation could be a consequence genetic, epigenetic, or environmental differences that affect the balance between telomere shortening and telomere elongation, such as processivity of telomerase and its expression levels, and the affinity of telomere-binding proteins to telomeric D N A (Blackburn, 2001). Superimposed over the bulk telomere length distributions were additional shorter telomeres that were reminiscent of the shortened telomeric outliers observed in human clonal fibroblast cultures (Baird et al., 2003). Although telomere length varies in different wild-type strains, the degree of this additional heterogeneity was similar in all strains except TR403, in which short outlying telomeres seemed to be more abundant. However, this abundance was likely to be caused by the long telomeres in the strain, so short outlying telomeres were preferably amplified. Because the degree of telomere length heterogeneity in different C. elegans strains was similar, it may not be generated randomly, but via some tightly controlled mechanism. It is interesting to note that TR403 has a high copy number of the transposable element Tel (Hodgkin and Doniach, 1997). A transposable element inserted close to the telomere might change the chromatin structure near the telomere and thereby affecting telomere homeostasis. While each strain has its characteristic telomere length distribution, single-worm S T E L A revealed telomere length heterogeneity among progeny of the same parent. Although most progeny had a similar telomere distribution, individuals whose telomere lengths were noticeably elongated or shortened relative to others were observed (Figure 4-2). The fluctuation in wild-type telomere length over the generations as shown in Figure 4-1 could be originated from such heterogeneity. 4.4.2 Presence of short outlying telomeres in wild-type strains In all of the wild-type strains characterized, occasional short outlying telomeres could be observed (Figure 4-4). Because telomerase adds telomeric repeats to only a fraction of telomeres in each cell cycle (Teixeira et al., 2004), these short telomeres could be explained if they escaped telomerase elongation through all the cellular divisions. However, trt-  106  I(ok410) lost on average between 100 and 150bp per generation (Table 4-3; Figure 4-6), and the short outlying telomeres were almost always more than a few hundred basepairs shorter than the average telomere length (Figure 4-7). Therefore, they were likely to be generated by mechanism(s) unrelated to the end replication problem. Similar large-scale loss of telomeric D N A has been documented in different organisms, mostly in mutant backgrounds (Lustig, 2003). However, there is evidence that such rapid loss of telomeric D N A also occurs in normal cells: occasional short outliers were observed by S T E L A in human clonal fibroblast cultures (Baird et al., 2003), and products of intrachromatid recombination, the mechanism speculated to be responsible for rapid telomere loss, have been detected by 2D gels in telomerase-expressing human skin fibroblast cell line (BJ/hTERT) (Wang et al., 2004). S T E L A in different wild-type C. elegans strains now provide an additional piece of evidence for the occurrence of rapid telomere loss in normal cells. 4.4.3 Reduced telomere length heterogeneity in telomerase mutant The major pathway for telomere maintenance in various organisms is telomerase (Cohn and Blackburn, 1995; Fitzgerald et al., 1996; Greider and Blackburn, 1985; Morin, 1989; Prowse et al., 1993). In C. elegans, microarray data showed that the most significant expression of trt-1 was found in isolated oocytes and reproductive stage adults, whereas none of the four laval stages or mixed-stage embryos gave significant expression (Hill et al., 2000). B y dividing the different stages of embryogenesis, it was reported that in fact trt-1 expression can be detected in embryos from certain stages (Baugh et al., 2003). Figure 4-3 shows that telomeres whose lengths were distinct from the majority were more abundant in animals that developed a functional germline than germlineless animals. Therefore, it appears that the germline may be an important source of telomere length heterogeneity. S T E L A demonstrated that trt-1 contributes to telomere length heterogeneity both within an animal and within a clonal population: V L telomere length distribution within an individual is narrower, and variations among siblings are much smaller in trt-1 (ok410) (Figure 4-7). It can be speculated that telomerase action in the germ cells is responsible for the observed variations among the clonal population, and telomerase action in the embryo could contribute to the heterogeneity seen within each individual.  107  TRT-1 is unusually small relative to the known telomerases from other species, and lacks some domains that are shared among the other telomerases (Malik et al., 2000; Figure 1-8). The progressive telomere shortening phenotype in trt-l(ok410) suggests that TRT-1 is indeed the reverse transcriptase component, as predicted based on its sequence (Malik et al., 2000). Decrease in telomere length heterogeneity has also been reported in the telomerase mutant strain in Arabidopsis, AtTERT-/- (Fitzerald et al., 1999; Riha et al., 2001, Heacock et al., 2004). While TRF analysis results in a smear in wild-type, discrete bands appear immediately after generation of the homozygous telomerase mutant (Fitzerald et al., 1999; Riha et al., 2001). The number of bands observed roughly correlates with the number of chromosome ends (ten chromosome ends from five chromosomes) (Fitzerald et al., 1999; Riha et al., 2001). Furthermore, PETRA, which uses probes specific for different chromosome ends, revealed a single discrete band for almost all chromosome ends examined (Heacock et al., 2004). These observations strongly suggest that each discrete band represents a particular chromosome end in both TRF analysis and P E T R A (Heacock et al., 2004). In wild-type Arabidopsis, each chromosome end has a wider telomere length distribution (Heacock et al., 2004), leading to a smear in TRF analysis. S. cerevisiae telomerase mutants also show a decrease in telomere length heterogeneity, which is manifested as a shorter smear in TRF analysis (Lundblad and Szostak, 1989; Lendvay et al., 1996; Lingner et al., 1997). Discrete bands were not observed, which is not surprising because of the relatively tight telomere distribution and the large number of chromosomes (16 chromosomes) in S. cerevisiae. Contrary to all the above organisms, telomere length heterogeneity appears to be similar between wild-type and the telomerase mutant in mouse in early generations (Blasco et al., 1997; Hande et al., 1999; Erdmann et al., 2004). This could again be attributed to the relatively large chromosome number in mouse (20 chromosomes). Telomere length analysis of individual chromosome ends may reveal reduction in telomere length heterogeneity in the mouse telomerase mutants. Telomere lengths of chromosomes 2p, 2q, 1 l p and 1 l q have been measured separately in cell lines derived from mTERT-/- (Hande et al., 1999). Interestingly, telomeres on some of these chromosome ends actually have increased heterogeneity (Hande et al., 1999), in contrary to telomerase mutants in S. cerevisiae, Arabidopsis, and C. elegans (Lundblad and Szostak, 1989; Lendvay et al., 1996; Lingner et al., 1997; Fitzerald et al., 1999; Riha et al., 2001, Heacock et al., 2004; this  108  chapter). However, the increase is mainly due to the appearance of many long telomeres, possibly indicating the emergence of a telomerase-independent telomere maintenance mechanism (Hande et al., 1999). Telomerase-independent telomere maintenance has been well documented in yeast and mammalian cell cultures; in both cases, telomere length becomes extremely heterogeneous (Teng and Zakian, 1999; Henson et al., 2002). 4.4.4 Increased frequency of short outlying telomeres in trt-1 (ok410) Besides progressively shortening telomeres and reduced telomere length heterogeneity, trtl(ok410) displayed an increased frequency of short outlying telomeres. This increase was apparent only when average telomere length was above ~lkb in the mutant (Compare different panels in Figure 4-7). The size difference between most of these short telomeres and the average telomeres suggests that they did not result from replicative loss. The model put forward initially by Lustig and colleagues to explain telomere rapid deletion in rapl' yeast mutants (Bucholc et al., 2001; Kyrion et al., 1992; L i and Lustig, 1996), and later by Wang et al. (2004) to account for the presence of T-loop-sized, telomeric repeat-containing circular D N A induced by T R F , involves intrachromatid recombination at telomeres. Similar AB  processes could generate the short outlying telomeres in trt-1 (ok410), and telomerase could counteract these processes in wild-type. Recent studies suggest that telomerase may protect chromosome ends in addition to elongating telomeres (Jacob et al., 2003; Masutomi et al., 2003; Taggart et al., 2002). Therefore, there are at least two possibilities for the increased frequency of short telomeres in trt-1: inability to elongate shortened telomeres, and/or lack of end protection by telomerase. Results of S T E L A suggest that there were at least two mechanisms of telomere shortening in trt-1 (ok410): replicative loss, which was the major contributor to the observed progressive shortening of average telomere length, and rapid telomere loss, which resulted in short outlying telomeres. In both mTert-/- and mTR-/- mice, Q-FISH revealed an abundance of telomeres with no detectable telomeric repeats in late generations or upon prolonged cell culturing, indicating the failure to elongate short telomere ends in the mutants (Hande et al., 1999; Hemann et al., 2001; Liu et al., 2002). This observation may be related to the high frequency of short outliers detected by S T E L A in trt-l(ok410). However, in those studies, the large spread of telomere lengths made it impossible to distinguish between replicative loss  109  and rapid telomere loss as the source of the signal-free ends. Furthermore, any change in the frequency of short outlying telomeres would be masked because of the large range of telomere lengths. 4.4.5 Rate of telomere shortening in trt-l(ok410) V L telomeres were found to shorten by, on average, 100 to 150 bp per generation in the absence of telomerase (Table 4-3 and Table 4-4). With an estimation of an average of 10 cell divisions per generation (Sulston and Horvitz, 1977; Sulston et al., 1983; Ahmed and Hodgkin, 2000), a cell loses approximately 10 to 15bp of telomeric D N A in C. elegans i f telomere loss is distributed evenly over each cell division. Although this rate was measured from only one chromosome end, it is expected that different chromosome ends would be comparable. In both human and mouse, the rate of telomere shortening was found to be between 100 and 150 bp per cell division in the absence of telomerase, while it is ~4bp in yeast (Counter et al., 1992; Harley et al., 1990; Lundblad and Szostak, 1989). Therefore, C. elegans may be more similar to yeast in terms of this rate of telomere loss. The difference in the rate of telomere shortening may reflect the difference in the length of the 3' overhang in various organisms. It is interesting to note that in mrt-2, telomeres are shortened at a similar rate as in trtl(ok410) (~125bp per generation, Ahmed & Hodgkin, 2000), suggesting that telomere shortening in mrt-2 could be caused by lack of elongation by telomerase. Ahmed and Hodgkin (2000) speculated that mrt-2, in its role as a checkpoint protein, could recognize telomeres as a form of D N A damage during replication, when telomeres are expected to adapt an open structure, revealing the double-stranded breaks (Blackburn, 2001). Mrt-2 could contribute to telomerase recruitment, possibly by coordinating the temporal and spatial association of telomerase. Despite the similar rate of telomere shortening in mrt-2 and trt-1, mrt-2 undergoes more generations before dying (between 12 and 22; Ahmed and Hodgkin, 2000). This could be explained by longer starting telomere length in the mrt-2 line examined by Ahmed and Hodgkin (2000). One major noticeable difference in telomere phenotype between mrt-2 and trt-1(ok410) is the lack of short outlying telomeres in mrt-2 (compare F2 in Figure 3-6 and Figure 4-7 panels A and B). Perhaps in mrt-2 telomerase is defective in  110  elongating telomeres after replication but it is still capable of protecting telomeres from undergoing rapid loss. 4.4.6 Absence of telomere defects in dog-l(gklO) It was proposed that dog-1 is required for resolving secondary structures formed by G-rich single-stranded D N A during lagging strand synthesis (Cheung et al., 2002). Because telomeric D N A is composed of G-rich repeats, it could also undergo sporadic deletions in dog-1 (gklO). To test the idea, two separate lines of dog-1 (gklO) were analyzed by S T E L A (Figure 4-7). If deletions did occur in telomeric D N A , telomeres could shorten over the generations, or there would be a higher frequency of short outlying telomeres. No obvious telomere defect was observed in dog-1 (gklO), indicating that dog-1 was not required for telomere maintenance and that deletions did not occur at a significant frequency in telomeric D N A in the absence of functional DOG-1.  Ill  C H A P T E R FIVE General discussion In this thesis, the mutator phenotype of a C. elegans strain mutated for the putative helicase gene dog-1, dog-1 (gklO) was investigated. The mutator phenotype was found to stem from a high frequency of deletions involving a very specific type of repeats, ones that are composed of long runs of uninterrupted guanines. A model in which formation of D N A secondary structures during lagging strand synthesis causes the observed unidirectional deletions in the mutants was discussed. Because telomeric D N A is a long tract of repeat sequences, signs of deletions within telomeric D N A were sought using S T E L A to measure telomere length in the mutants. No significant difference between them and wild-type was observed. S T E L A also provided a unique opportunity to examine individual difference in telomere length. Telomere length heterogeneity among individuals within a clonal population was noted. This heterogeneity was reduced when telomerase was defective. In addition, S T E L A allowed detection of rare large-scale loss of telomeric D N A in wild-type, a phenomenon that became more pronounced in telomerase mutants. 5.1 Relation of DOG-1 to BACH1 The best match of DOG-1 in human is BACH1 (David L. Baillie, personal communication), a helicase that interacts with BRCA1 and has been implicated in checkpoint control (Cantor et al., 2001; Cantor et al., 2004; Y u et al., 2003; Rodriguez et al., 2003). Interaction between B R C A 1 and B A C H 1 depends on phosphorylation of the C-terminal domain of B A C H 1 (Yu et al., 2003; Rodriguez et al., 2003). DOG-1 lacks this C-terminal domain of B A C H 1 , suggesting that DOG-1 does not carry out all functions of B A C H 1. Other than the C-terminal domain, the helicase domain of B A C H 1 is highly similar to DOG-1 (Figure 2-2). Similar to BACH1/BRIP1, DOG-1 appears to be required for D N A repair, and studies in dog-1 (gklO) suggested that this D N A repair is targeted towards a specific type of D N A damage, one that involves (G/C)n (Cheung et al., 2002). It is tempting to postulate that the observed genomic instability in dog-1 (gklO) is resulted from deficiency of its helicase activity. However, no biochemical evidence that DOG-1 is indeed a helicase has been obtained so far. Inhibition of B A C H 1 expression by siRNA results in defects in the G 2 / M checkpoint, and expression of B A C H 1 mutants harboring missense mutations that abolish interaction with B R C A 1 fails to rescue the checkpoint defect, suggesting that it is the interaction  112  between B R C A 1 and B A C H 1 , rather than the helicase activity, that is important for B A C H 1 's checkpoint function (Yu et a l , 2003; Rodriquez et al., 2003). Therefore, it appears that B A C H 1 may be divided into two functionally separable domains: the helicase domain is required for D N A repair, and the C-terminal domain is responsible for G 2 / M checkpoint function through its interaction with BRCA1 (Yu et al., 2003; Rodriquez et al., 2003). Because DOG-1 lacks the C-terminal domain found in BACH1/BRIP1, DOG-1 may not have a cell cycle checkpoint function. In this case, a different mechanism may be employed in slowing down the cell cycle while repair takes place. Dog-l(gklO) is defective in the maintenance of (G/C)n specifically. There has been no documentation on the types of D N A damage that B A C H 1 acts on. It will be interesting to see if BACH1-/- cells also display a high frequency of deletions involving long (G/C)n.  5.2 Absence of significant telomere defects in dog-l(gklO) Telomeric D N A is in general G-rich and the G-strands from ciliate and human telomeres have been shown to form G-quadruplexes in vitro (Williamson et al., 1989; Sundquist and Klug, 1989; Kang et al., 1992; Smith and Feigon, 1992; Wang and Petel, 1993; Parkinson et al., 1993). Therefore, the rationale for examining telomere length in dog-l(gklO) was that telomeric D N A may also form secondary structures similar to (G/C)n during replication, because telomeric D N A is a very long repeat tract relative to the (G/C)n that were involved in deletions, and so the likelihood of secondary structure formation should be higher. Most importantly, the G-strand of telomeric D N A is replicated by lagging strand synthesis. Formation of secondary structures by telomeric D N A in dog-1 (gklO) is predicted to result in sporadic deletions, which could be manifested as increased frequency of short outliers and/or telomere shortening. Nevertheless, the absence of detectable telomere defects in dog-1 (gklO) as analyzed by S T E L A is not entirely surprising. Examination of other repeat types in dog-1 (gklO) showed that a long run of uninterrupted guanines is required for deletions to occur most of the time. Based on existing knowledge, the most probable structure that would only be formed by long (G)n is a G-quadruplex. Presumably long (G)n allows formation of more stable G-quadruplexes with many G-quartet planes. It is also possible that long (G)n enables formation of different G-quadruplexes (in other words, there may be more than one way a G 113  quadruple* could form from a long (G)n), thus increasing the probability of one forming. It should be noted that (CGG)n, which has been shown to form G-quadruplexes in vitro (Fry and Loeb, 1994), was stable in dog-1(gklO). It is possible that the stability of (CGG)n is due to their relatively short tract lengths: the longest (CGG)n tracts examined were only 21 nt in length. Furthermore, such relatively short tracts are likely to restrict how a G-quadruplex could form, thus lowering the probability of its formation. In C. elegans, the telomeric repeat is T T A G G C (Wicky et al., 1996). Therefore, although telomeric D N A is relatively long, a G-quadruplex formed by T T A G G C repeats would only be stabilized by a stack of two G-quartet planes. The structure could potentially involve two or more stacks of two G-quartet planes, but the intervening space may be destabilizing. A l l telomeric repeats that have been shown to form G-quadruplexes in vitro contain at least three consecutive guanines (e.g. T T A G G G in vertebrates). Hence, i f a G quadruplex is formed by C. elegans telomeric D N A , it may not be stable enough to cause deletions in dog-l(gklO).  5.3 Recommendations and considerations in using S T E L A The experiments described in Chapters 3 and 4 demonstrate the usefulness of S T E L A in studying telomere biology in C. elegans. Its ability to measure telomere length of a particular telomere end from single worms means it allows rigorous comparisons among different strains. It eliminates two levels of telomere length variations: individual difference in telomere length, and variations among different chromosome ends. The technique has also revealed rare long or short outlying telomeres that can become more or less abundant in different genetic backgrounds. The observation of rare short outlying telomeres in wild-type is evidence of stochastic events that shorten telomeres to different extents. The frequency of these events could be measured to study their nature. It is believed that critically short telomeres, instead of short average telomere length, cause the cell to senesce (Karlseder et al., 2002; L i et al., 2003; Stewart et al., 2003). Therefore, the ability to detect short outliers in C. elegans will enable it to become a new model for studying the phenomenon. Two considerations in analyzing telomeres by S T E L A are discussed below: The amount of D N A template used in each PCR reaction could be adjusted to suit the purposes for different experiments. Carrying out PCR at an amount of D N A that allows  114  separation of discrete bands permits quantification of bands with different lengths. Multiple reactions could be set up to increase significance. However, i f one had a large number of samples (for example, in performing a large scale screen for a particular telomere phenotype), setting up multiple reactions for each sample could become tedious. Therefore, in cases where quantification is unnecessary or average telomere length is the major parameter of concern, more D N A could be used in each reaction, and one or few reactions would be needed for each sample. The disadvantage of doing this is that subtle changes to telomere distribution could be overlooked. For instance, results in Chapter 4 and Baird et al. (2002) revealed bands that were significantly different in size than the bulk population. Because of the rarity of these bands, including too much template D N A in a reaction could lower the sensitivity of S T E L A in detecting these bands. In initial S T E L A experiments, a few worms were sampled from the population, D N A extracted, and 4 to 8 PCR reactions were set up for each sample (Figures 3-4 to 3-6, and data not shown). In later experiments, it was realized that there could be substantial variations in telomere length even within a clonal population (Figure 4-2). Therefore, while sampling a number of worms could be considered to be a representation of the population, it would mask any individual variations, and also reduce the resolution of STELA. Hence, for some cases, carrying out S T E L A separately on a number of individuals sampled randomly from a population may be more informative. If quantification of bands of different sizes is not required, using the whole ligated genomic D N A from an individual worm in a reaction would not usually compromise the sensitivity of STELA. Heacock et al. (2004) also devised a PCR-based method for measuring telomere length in Arabidopsis. Briefly, an adaptor primer, termed PETRA-T, is first hybridized to the 3' overhang (Heacock et al., 2004). PETRA-T contains twelve nucleotides complementary to the telomeric G-strand at its 3' end, and a unique sequence tag at its 5' end (Heacock et al., 2004). After annealing, primer extension is carried out by D N A Poll. The 5' —> 3' exonuclease activity of the polymerase enables the enzyme to replace nucleotides in the growing strand of D N A by nick translation. As a result, the primer extension step synthesizes a new D N A strand that has the unique sequence tag at its 5' end (Heacock et al., 2004). This newly synthesized strand is then used as a template for PCR using a unique subtelomeric  115  primer, and a primer (PETRA-A) whose sequence is identical to the tag on PETRA-T (Heacock et al., 2004; Figure 3-7). Both S T E L A and PETRA take advantage of the presence of a 3' overhang to anneal an oligonucleotide to telomere termini, and both use a unique subtelomeric primer to allow amplification of a specific chromosome end. However, there are a number of distinctions between the two techniques. First, annealing of the oligonucleotide to the telomeric overhang is followed by ligation to the 5' end of the telomere (i.e. the C-strand) in S T E L A , while it is followed by primer extension to replace the entire strand carrying the telomeric C-strand in PETRA. Second, because the oligonucleotide is ligated to the 5' end of the telomere, S T E L A essentially measures the length of the telomeric C-strand. In PETRA, primer extension extends the oligonucleotide annealed to the 3' most end of the overhang; therefore, P E T R A essentially measures the length of the telomeric G-strand. S T E L A in both human cells (Baird et al., 2003) and C. elegans (chapter four) revealed the presence of occasional short outlying telomeres in normal cells. Such outlying telomeres are not evident in Heacock et al. (2004) in either wild-type or mutant background, indicating that these events may be extremely rare in Arabidopsis that they could not be detected even by the PCR-based technique. This is unexpected because they have been observed in both human (Baird et al., 2003) and C. elegans (Cheung et al., 2004; Chapter 4), suggesting that they arise from certain conserved processes at the telomere.  116  Annealing of the PETRA-T oiigo to a 3' G-overhang  A  HtlllllllHIIIIIUIII Primer extension with DNAPoflat25 C a  A  _3'  Synthesis of the G-strand with adaptor during the first PCR cycle  It Amplification of a telomere by PCR using a subtelomeric and PETRA-A ollgos  l l l l h l l l 11 l I l l l l l l M l l l l i Figure 5-1. Principle of P E T R A in telomere length measurement mArabidposis. adapted from Heacock et al. (2004).  117  (Figure  BIBLIOGRAPHY  Aaltonen, L . A . , Peltomaki, P., Leach, F. S., Sistonen, P., Pylkkanen, L., Mecklin, J. P., Jarvinen, H . , Powell, S. M . , Jen, J., Hamilton, S. R., and et al. (1993). Clues to the pathogenesis of familial colorectal cancer. Science 260, 812-816. Ahmed, S., Alpi, A., Hengartner, M . O., and Gartner, A . (2001). C. elegans RAD-5/CLK-2 defines a new D N A damage checkpoint protein. Curr Biol 11, 1934-1944. Ahmed, S., and Hodgkin, J. (2000). MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature 403, 159-164. Ambrus, A . , Chen, D., Dai, J., Jones, R. A., and Yang, D. (2005). Solution Structure of the Biologically Relevant G-Quadruplex Element in the Human c - M Y C Promoter. Implications for G-Quadruplex Stabilization. Biochemistry 44, 2048-2058. Armbruster, B. N . , Banik, S. S., Guo, C , Smith, A . C , and Counter, C. M . (2001). N terminal domains of the human telomerase catalytic subunit required for enzyme activity in vivo. M o l Cell Biol 21, 7775-7786. Armbruster, B. N . , Etheridge, K . T., Broccoli, D., and Counter, C. M . (2003). Putative telomere-recruiting domain in the catalytic subunit of human telomerase. M o l Cell Biol 23, 3237-3246. Bachand, F., and Autexier, C. (2001). Functional regions of human telomerase reverse transcriptase and human telomerase R N A required for telomerase activity and RNA-protein interactions. M o l Cell Biol 21, 1888-1897. Bachrati, C. Z., and Hickson, I. D. (2003). RecQ helicases: suppressors of tumorigenesis and premature aging. Biochem J 374, 577-606. Bailey, S. M . , Cornforth, M . N . , Kurimasa, A . , Chen, D. J., and Goodwin, E. H . (2001). Strand-specific postreplicative processing of mammalian telomeres. Science 293, 2462-2465. Baird, D. M . , Rowson, J., Wynford-Thomas, D., and Kipling, D. (2003). Extensive allelic variation and ultrashort telomeres in senescent human cells. Nat Genet 33, 203-207. Baker, T. A . , and Bell, S. P. (1998). Polymerases and the replisome: machines within machines. Cell 92,295-305.  118  Balakumaran, B . S., Freudenreich, C. H., and Zakian, V . A . (2000). C G G / C C G repeats exhibit orientation-dependent instability and orientation-independent fragility in Saccharomyces cerevisiae. Hum Mol Genet 9, 93-100. Baran, N . , Lapidot, A., and Manor, H. (1991). Formation of D N A triplexes accounts for arrests of D N A synthesis at d(TC)n and d(GA)n tracts. Proc Natl Acad Sci U S A 88, 507511. Baran, N . , Pucshansky, L., Marco, Y . , Benjamin, S., and Manor, H . (1997). The SV40 large T-antigen helicase can unwind four stranded D N A structures linked by G-quartets. Nucleic Acids Res 25, 297-303. Barnes, T. M . , Kohara, Y . , Coulson, A., and Hekimi, S. (1995). Meiotic recombination, noncoding D N A and genomic organization in Caenorhabditis elegans. Genetics 141,159179. Bass, H . W. (2003). Telomere dynamics unique to meiotic prophase: formation and significance of the bouquet. Cell Mol Life Sci 60, 2319-2324. Baugh, L. R., Hill, A . A., Slonim, D. K., Brown, E. L., and Hunter, C. P. (2003). Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome. Development 130, 889-900. Baumann, P., and Cech, T. R. (2001). Potl, the putative telomere end-binding protein in fission yeast and humans. Science 292, 1171-1175. Beanan, M . J., and Strome, S. (1992). Characterization of a germ-line proliferation mutation in C. elegans. Development 116, 755-766. Bekaert, S., Derradji, H., and Baatout, S. (2004). Telomere biology in mammalian germ cells and during development. Dev Biol 274, 15-30. Benard, C , McCright, B., Zhang, Y . , Felkai, S., Lakowski, B., and Hekimi, S. (2001). The C. elegans maternal-effect gene clk-2 is essential for embryonic development, encodes a protein homologous to yeast Tel2p and affects telomere length. Development 128, 4045-4055. Benkovic, S. J., Valentine, A . M . , and Salinas, F. (2001). Replisome-mediated D N A replication. Annu Rev Biochem 70,181-208. Berry, L. W., Westlund, B., and Schedl, T. (1997). Germ-line tumor formation caused by activation of glp-1, a Caenorhabditis elegans member of the Notch family of receptors. Development 124, 925-936.  119  Bichara, M . , Pinet, I., Schumacher, S., and Fuchs, R. P. (2000). Mechanisms of dinucleotide repeat instability in Escherichia coli. Genetics 154, 533-542. Bichara, M . , Schumacher, S., and Fuchs, R. P. (1995). Genetic instability within monotonous runs of CpG sequences in Escherichia coli. Genetics 140, 897-907. Blackburn, E. H . (2001). Switching and signaling at the telomere. Cell 106, 661-673. Blackburn, E. H., and Gall, J. G. (1978). A tandemly repeated sequence at the termini of the extrachromosomal ribosomal R N A genes in Tetrahymena. J M o l Biol 720, 33-53. Blander, G., Kipnis, J., Leal, J. F., Y u , C. E., Schellenberg, G. D., and Oren, M . (1999). Physical and functional interaction between p53 and the Werner's syndrome protein. J Biol Chem 274, 29463-29469. Blasco, M . A., Lee, H . W., Hande, M . P., Samper, E., Lansdorp, P. M . , DePinho, R. A . , and Greider, C. W. (1997). Telomere shortening and tumor formation by mouse cells lacking telomerase R N A . Cell 91, 25-34. Boulton, S. J., Martin, J. S., Polanowska, J., Hill, D. E., Gartner, A., and Vidal, M . (2004). B R C A 1 / B A R D 1 orthologs required for D N A repair in Caenorhabditis elegans. Curr Biol 14, 33-39. Boyer, J. C , Yamada, N . A., Roques, C. N . , Hatch, S. B., Riess, K., and Farber, R. A . (2002). Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells. Hum Mol Genet 11, 707-713. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94. Broccoli, D., Smogorzewska, A., Chong, L., and de Lange, T. (1997). Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2. Nat Genet 17, 231-235. Broccoli, D., Young, J. W., and de Lange, T. (1995). Telomerase activity in normal and malignant hematopoietic cells. Proc Natl Acad Sci U S A 92, 9082-9086. Brosh, R. M . , Jr., L i , J. L., Kenny, M . K., Karow, J. K., Cooper, M . P., Kureekattil, R. P., Hickson, I. D., and Bohr, V . A . (2000). Replication protein A physically interacts with the Bloom's syndrome protein and stimulates its helicase activity. J Biol Chem 275, 2350023508. Brosh, R. M . , Jr., and Matson, S. W. (1995). Mutations in motif II of Escherichia coli D N A helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction. J Bacteriol 177, 5612-5621.  120  Brosh, R. M . , Jr., Orren, D. K . , Nehlin, J. O., Ravn, P. H., Kenny, M . K., Machwe, A . , and Bohr, V . A . (1999). Functional and physical interaction between W R N helicase and human replication protein A . J Biol Chem 274,18341-18350. Brosh, R. M . , Jr., von Kobbe, C , Sommers, J. A., Karmakar, P., Opresko, P. L., Piotrowski, J., Dianova, I., Dianov, G. L., and Bohr, V . A . (2001). Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity. E M B O J 20, 5791-5801. Bryan, T. M . , Goodrich, K . J., and Cech, T. R. (2000a). A mutant of Tetrahymena telomerase reverse transcriptase with increased processivity. J Biol Chem 275, 24199-24207. Bryan, T. M . , Goodrich, K . J., and Cech, T. R. (2000b). Telomerase R N A bound by protein motifs specific to telomerase reverse transcriptase. Mol Cell 6, 493-499. Bucholc, M . , Park, Y . , and Lustig, A . J. (2001). Intrachromatid excision of telomeric D N A as a mechanism for telomere size control in Saccharomyces cerevisiae. M o l Cell Biol 21, 65596573. Cantor, S., Drapkin, R., Zhang, F., Lin, Y . , Han, J., Pamidi, S., and Livingston, D. M . (2004). The BRCA1-associated protein BACH1 is a D N A helicase targeted by clinically relevant inactivating mutations. Proc Natl Acad Sci U S A 101, 2357-2362. Cantor, S. B., Bell, D. W., Ganesan, S., Kass, E. M . , Drapkin, R., Grossman, S., Wahrer, D. C , Sgroi, D. C , Lane, W. S., Haber, D. A., and Livingston, D. M . (2001). B A C H 1 , a novel helicase-like protein, interacts directly with BRCA1 and contributes to its D N A repair function. Cell 105, 149-160. Catasti, P., Chen, X . , Moyzis, R. K . , Bradbury, E. M . , and Gupta, G. (1996). Structurefunction correlations of the insulin-linked polymorphic region. J Mol Biol 264, 534-545. Cesare, A . J., Quinney, N . , Willcox, S., Subramanian, D., and Griffith, J. D. (2003). Telomere looping in P. sativum (common garden pea). Plant J 36,271-279. Chen, J. L., Blasco, M . A . , and Greider, C. W. (2000). Secondary structure of vertebrate telomerase R N A . Cell 100, 503-514. Chen, J. L., and Greider, C. W. (2004). A n emerging consensus for telomerase R N A structure. Proc Natl Acad Sci U S A 101, 14683-14684. Cheung, I., Schertzer, M . , Baross, A., Rose, A . M . , Lansdorp, P. M . , and Baird, D. M . (2004). Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans. Nucleic Acids Res 32, 3383-3391.  121  Cheung, I., Schertzer, M . , Rose, A . , and Lansdorp, P. M . (2002). Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich D N A . Nat Genet 31, 405409. Chong, L., van Steensel, B., Broccoli, D., Erdjument-Bromage, FL, Hanish, J., Tempst, P., and de Lange, T. (1995a). A human telomeric protein. Science 270,1663-1667. Chong, S. S., McCall, A . E., Cota, J., Subramony, S. H., Orr, H . T., Hughes, M . R., and Zoghbi, H . Y . (1995b). Gametic and somatic tissue-specific heterogeneity of the expanded SCA1 C A G repeat in spinocerebellar ataxia type 1. Nat Genet 10, 344-350. Chung, M . Y . , Ranum, L. P., Duvick, L. A . , Servadio, A., Zoghbi, H . Y . , and Orr, H . T. (1993). Evidence for a mechanism predisposing to intergenerational C A G repeat instability in spinocerebellar ataxia type I. Nat Genet 5, 254-258. Clapperton, J. A . , Manke, I. A . , Lowery, D. M . , Ho, T., Haire, L. F., Yaffe, M . B., and Smerdon, S. J. (2004). Structure and mechanism of BRCA1 B R C T domain recognition of phosphorylated B A C H 1 with implications for cancer. Nat Struct M o l Biol 11, 512-518. Colin, M . , and Blackburn, E. H . (1995). Telomerase in yeast. Science 269, 396-400. Colgin, L., and Reddel, R. (2004). Telomere biology: a new player in the end zone. Curr Biol 14, R901-902. Collins, K . (1999). Ciliate telomerase biochemistry. Annu Rev Biochem 68, 187-218. Conrad, M . N . , Wright, J. H., Wolf, A . J., and Zakian, V . A . (1990). RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability. Cell 63, 739-750. Constantinou, A . , Tarsounas, M . , Karow, J. K., Brosh, R. M . , Bohr, V . A . , Hickson, I. D., and West, S. C. (2000). Werner's syndrome protein (WRN) migrates Holliday junctions and co-localizes with R P A upon replication arrest. E M B O Rep 1, 80-84. Cooper, J. P., Nimmo, E. R., Allshire, R. C , and Cech, T. R. (1997). Regulation of telomere length and function by a Myb-domain protein in fission yeast. Nature 385,144-141. Cooper, M . P., Machwe, A . , Orren, D. K., Brosh, R. M . , Ramsden, D., and Bohr, V . A . (2000). K u complex interacts with and stimulates the Werner protein. Genes Dev 14, 907912.  122  Counter, C. M . , Avilion, A . A., LeFeuvre, C. E., Stewart, N . G., Greider, C. W., Harley, C. B., and Bacchetti, S. (1992). Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. E M B O J 11, 1921-1929. Counter, C. M . , Meyerson, M . , Eaton, E. N . , and Weinberg, R. A . (1997). The catalytic subunit of yeast telomerase. Proc Natl Acad Sci U S A 94, 9202-9207. Coviello-McLaughlin, G. M . , and Prowse, K . R. (1997). Telomere length regulation during postnatal development and ageing in Mus spretus. Nucleic Acids Res 25, 3051-3058. Cummings, C. J., and Zoghbi, H . Y . (2000). Fourteen and counting: unraveling trinucleotide repeat diseases. Hum M o l Genet 9, 909-916. Dalgaard, J. Z., and Klar, A . J. (2001). A D N A replication-arrest site RTS1 regulates imprinting by determining the direction of replication at matl in S. pombe. Genes Dev 15, 2060-2068. de Lange, T. (2002). Protection of mammalian telomeres. Oncogene 21, 532-540. de Lange, T., Shiue, L., Myers, R. M . , Cox, D. R., Naylor, S. L., Killery, A . M . , and Varmus, H . E. (1990). Structure and variability of human chromosome ends. M o l Cell Biol 10, 518527. Dechering, K . J., Cuelenaere, K., Konings, R. N . , and Leunissen, J. A . (1998). Distinct frequency-distributions of homopolymeric D N A tracts in different genomes. Nucleic Acids Res 26, 4056-4062. Degtyareva, N . P., Greenwell, P., Hofmann, E. R., Hengartner, M . O., Zhang, L., Culotti, J. G., and Petes, T. D. (2002). Caenorhabditis elegans D N A mismatch repair gene msh-2 is required for microsatellite stability and maintenance of genome integrity. Proc Natl Acad Sci U S A 99, 2158-2163. Dempsey, L. A., Hanakahi, L. A., and Maizels, N . (1998). A specific isoform of hnRNP D interacts with D N A in the LR1 heterodimer: canonical R N A binding motifs in a sequencespecific duplex D N A binding protein. J Biol Chem 273, 29224-29229. Dempsey, L. A., Sun, H., Hanakahi, L. A., and Maizels, N . (1999). G4 D N A binding by LR1 and its subunits, nucleolin and hnRNP D, A role for G-G pairing in immunoglobulin switch recombination. J Biol Chem 274,1066-1071. Denver, D. R., Morris, K., Kewalramani, A., Harris, K. E., Chow, A., Estes, S., Lynch, M . , and Thomas, W. K . (2004a). Abundance, distribution, and mutation rates of homopolymeric nucleotide runs in the genome of Caenorhabditis elegans. J Mol Evol 58, 584-595.  123  Denver, D . R., Morris, K., Lynch, M . , and Thomas, W. K . (2004b). High mutation rate and predominance of insertions in the Caenorhabditis elegans nuclear genome. Nature 430, 679682. Dieringer, D., and Schlotterer, C. (2003). Two distinct modes of microsatellite mutation processes: evidence from the complete genomic sequences of nine species. Genome Res 13, 2242-2251. Ding, H., Schertzer, M . , Wu, X . , Gertsenstein, M . , Selig, S., Kammori, M . , Pourvali, R., Poon, S., Vulto, I., Chavez, E., et al. (2004). Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein. Cell 117, 873-886. Drake, J. W., Charlesworth, B., Charlesworth, D., and Crow, J. F. (1998). Rates of spontaneous mutation. Genetics 148,1667-1686. Duquette, M . L., Handa, P., Vincent, J. A., Taylor, A . F., and Maizels, N . (2004). Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 D N A . Genes Dev 18, 1618-1629. Eichler, E. E., Holden, J. J., Popovich, B. W., Reiss, A . L., Snow, K., Thibodeau, S. N . , Richards, C. S., Ward, P. A., and Nelson, D. L . (1994). Length of uninterrupted C G G repeats determines instability in the FMR1 gene. Nat Genet 8, 88-94. Eickbush, T. H . (1997). Telomerase and retrotransposons: which came first? Science 277, 911-912. Ellegren, H . (2000). Microsatellite mutations in the germline: implications for evolutionary inference. Trends Genet 16, 551-558. Erdmann, N . , Liu, Y . , and Harrington, L. (2004). Distinct dosage requirements for the maintenance of long and short telomeres in mTert heterozygous mice. Proc Natl Acad Sci U S A 101, 6080-6085. Evans, E., Moggs, J. G., Hwang, J. R., Egly, J. M . , and Wood, R. D. (1997). Mechanism of open complex and dual incision formation by human nucleotide excision repair factors. E M B O J 16, 6559-6573. Evans, S. K., and Lundblad, V . (1999). Estl and Cdcl3 as comediators of telomerase access. Science 286,117-120. Fang, G., and Cech, T. R. (1993a). The beta subunit of Oxytricha telomere-binding protein promotes G-quartet formation by telomeric D N A . Cell 74, 875-885.  124  Fang, G., and Cech, T. R. (1993b). Oxytricha telomere-binding protein: DNA-dependent dimerization of the alpha and beta subunits. Proc Natl Acad Sci U S A 90, 6056-6060. Ferreira, M . G., and Cooper, J. P. (2001). The fission yeast Tazl protein protects chromosomes from Ku-dependent end-to-end fusions. Mol Cell 7, 55-63. Field, D., and Wills, C. (1998). Abundant microsatellite polymorphism in Saccharomyces cerevisiae, and the different distributions of microsatellites in eight prokaryotes and S. cerevisiae, result from strong mutation pressures and a variety of selective forces. Proc Natl Acad Sci U S A 95, 1647-1652. Fire, A., X u , S., Montgomery, M . K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded R N A in Caenorhabditis elegans. Nature 391, 806-811. Fitzgerald, M . S., McKnight, T. D., and Shippen, D. E. (1996). Characterization and developmental patterns of telomerase expression in plants. Proc Natl Acad Sci U S A 93, 14422-14427. Fitzgerald, M . S., Riha, K., Gao, F., Ren, S., McKnight, T. D., and Shippen, D. E. (1999). Disruption of the telomerase catalytic subunit gene from Arabidopsis inactivates telomerase and leads to a slow loss of telomeric D N A . Proc Natl Acad Sci U S A 96,14813-14818. Flores-Rozas, H., and Kolodner, R. D. (1998). The Saccharomyces cerevisiae M L H 3 gene functions in MSH3-dependent suppression of frameshift mutations. Proc Natl Acad Sci U S A 95, 12404-12409. Ford, D., Easton, D. F., Stratton, M . , Narod, S., Goldgar, D., Devilee, P., Bishop, D. T., Weber, B., Lenoir, G., Chang-Claude, J., et al. (1998). Genetic heterogeneity and penetrance analysis of the BRCA1 and B R C A 2 genes in breast cancer families. The Breast Cancer Linkage Consortium. A m J Hum Genet 62, 676-689. Freudenreich, C. H., Kantrow, S. M . , and Zakian, V . A . (1998). Expansion and lengthdependent fragility of C T G repeats in yeast. Science 279, 853-856. Freudenreich, C. H., Stavenhagen, J. B., and Zakian, V . A . (1997). Stability of a C T G / C A G trinucleotide repeat in yeast is dependent on its orientation in the genome. M o l Cell Biol 17, 2090-2098. Friedman, K. L., and Cech, T. R. (1999). Essential functions of amino-terminal domains in the yeast telomerase catalytic subunit revealed by selection for viable mutants. Genes Dev 13, 2863-2874.  125  Fry, M . , and Loeb, L. A . (1994). The fragile X syndrome d(CGG)n nucleotide repeats form a stable tetrahelical structure. Proc Natl Acad Sci U S A 91, 4950-4954. Fry, M . , and Loeb, L . A . (1999). Human werner syndrome D N A helicase unwinds tetrahelical structures of the fragile X syndrome repeat sequence d(CGG)n. J Biol Chem 274, 12797-12802. Garvik, B., Carson, M . , and Hartwell, L . (1995). Single-stranded D N A arising at telomeres in cdcl3 mutants may constitute a specific signal for the RAD9 checkpoint. M o l Cell Biol 75, 6128-6138. Gellert, M . , Lipsett, M . N . , and Davies, D. R. (1962). Helix formation by guanylic acid. Proc Natl Acad Sci U S A 48, 2013-2018. George, S. E., Simokat, K., Hardin, J., and Chisholm, A . D. (1998). The V A B - 1 Eph receptor tyrosine kinase functions in neural and epithelial morphogenesis in C. elegans. Cell 92, 633643. Gilbert, D. E., and Feigon, J. (1999). Multistranded D N A structures. Curr Opin Struct Biol 9, 305-314. Gilley, D., and Blackburn, E. H . (1999). The telomerase R N A pseudoknot is critical for the stable assembly of a catalytically active ribonucleoprotein. Proc Natl Acad Sci U S A 96, 6621-6625. Gilson, E., Roberge, M . , Giraldo, R., Rhodes, D., and Gasser, S. M . (1993). Distortion of the D N A double helix by RAP1 at silencers and multiple telomeric binding sites. J M o l Biol 231, 293-310. Giraldo, R., and Rhodes, D. (1994). The yeast telomere-binding protein RAP1 binds to and promotes the formation of D N A quadruplexes in telomeric D N A . E M B O J 13, 2411-2420. Goetz, G. S., Dean, F. B., Hurwitz, J., and Matson, S. W. (1988). The unwinding of duplex regions in D N A by the simian virus 40 large tumor antigen-associated D N A helicase activity. J Biol Chem 263, 383-392. Gorbalenya, A . E., and Koonin, E. V . (1993). Helicases: amino acid sequence comparisons and structure-function relationships. Curr Opin Struct Biol 3,419-429. Gottschling, D. E., and Cech, T. R. (1984). Chromatin structure of the molecular ends of Oxytricha macronuclear D N A : phased nucleosomes and a telomeric complex. Cell 38, 501510.  126  Gottschling, D. E., and Zakian, V . A . (1986). Telomere proteins: specific recognition and protection of the natural termini of Oxytricha macronuclear D N A . Cell 47, 195-205. Gragg, H . , Harfe, B. D., and Jinks-Robertson, S. (2002). Base composition of mononucleotide runs affects D N A polymerase slippage and removal of frameshift intermediates by mismatch repair in Saccharomyces cerevisiae. Mol Cell Biol 22, 8756-8762. Grandin, N . , Damon, C , and Charbonneau, M . (2001). Tenl functions in telomere end protection and length regulation in association with Stnl and Cdcl3. E M B O J 20, 11731183. Grandin, N . , Reed, S. I., and Charbonneau, M . (1997). Stnl, a new Saccharomyces cerevisiae protein, is implicated in telomere size regulation in association with Cdcl3. Genes Dev 11, 512-527. Gray, M . D., Shen, J. C , Kamath-Loeb, A . S., Blank, A., Sopher, B. L., Martin, G. M . , Oshima, J., and Loeb, L. A . (1997). The Werner syndrome protein is a D N A helicase. Nat Genet 7 7, 100-103. Greider, C. W. (1998). Telomerase activity, cell proliferation, and cancer. Proc Natl Acad Sci U S A 95, 90-92. Greider, C. W., and Blackburn, E. H . (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405-413. Greider, C. W., and Blackburn, E. H . (1987). The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 51, 887-898. Greider, C. W., and Blackburn, E. H . (1989). A telomeric sequence in the R N A of Tetrahymena telomerase required for telomere repeat synthesis. Nature 337, 331-337. Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M . , Bianchi, A . , Moss, H . , and de Lange, T. (1999). Mammalian telomeres end in a large duplex loop. Cell 97, 503-514. Hackett, J. A . , Feldser, D. M . , and Greider, C. W. (2001). Telomere dysfunction increases mutation rate and genomic instability. Cell 106, 275-286. Haering, C . H . , Nakamura, T. M . , Baumann, P., and Cech, T. R. (2000). Analysis of telomerase catalytic subunit mutants in vivo and in vitro in Schizosaccharomycespombe. Proc Natl Acad Sci U S A 97, 6367-6372.  127  Hall, M . C , and Matson, S. W. (1999). Helicase motifs: the engine that powers D N A unwinding. M o l Microbiol 34, 867-877. Hamada, H., Seidman, M . , Howard, B . H., and Gorman, C. M . (1984). Enhanced gene expression by the poly(dT-dG).poly(dC-dA) sequence. Mol Cell Biol 4, leil-ie^Q. Hammond-Kosack, M . C , and Docherty, K . (1992). A consensus repeat sequence from the human insulin gene linked polymorphic region adopts multiple quadriplex D N A structures in vitro. FEBS Lett 301, 79-82. Hanakahi, L . A., and Maizels, N . (2000). Transcriptional activation by LR1 at the Emu enhancer and switch region sites. Nucleic Acids Res 28, 2651-2657. Hanakahi, L . A., Sun, H., and Maizels, N . (1999). High affinity interactions of nucleolin with G-G-paired rDNA. J Biol Chem 274, 15908-15912. Hande, M . P., Samper, E., Lansdorp, P., and Blasco, M . A . (1999). Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice. J Cell Biol 144, 589-601. Hanvey, J. C , Shimizu, M . , and Wells, R. D. (1988). Intramolecular D N A triplexes in supercoiled plasmids. Proc Natl Acad Sci U S A 85, 6292-6296. Hao, L. Y . , and Greider, C. W. (2004). Genomic instability in both wild-type and telomerase null MEFs. Chromosoma 113, 62-68. Hardy, C. F., Sussel, L., and Shore, D. (1992). A RAP 1-interacting protein involved in transcriptional silencing and telomere length regulation. Genes Dev 6, 801-814. Harfe, B . D., and Jinks-Robertson, S. (2000). Sequence composition and context effects on the generation and repair of frameshift intermediates in mononucleotide runs in Saccharomyces cerevisiae. Genetics 156, 571-578. Harfe, B . D., Minesinger, B . K., and Jinks-Robertson, S. (2000). Discrete in vivo roles for the MutL homologs Mlh2p and Mlh3p in the removal of frameshift intermediates in budding yeast. Curr Biol 10, 145-148. Harley, C. B . (1991). Telomere loss: mitotic clock or genetic time bomb? Mutat Res 256, 271-282. Harley, C. B., Futcher, A . B., and Greider, C. W. (1990). Telomeres shorten during ageing of human fibroblasts. Nature 345, 458-460.  128  Hastie, N . D., Dempster, M . , Dunlop, M . G., Thompson, A . M . , Green, D. K., and Allshire, R. C. (1990). Telomere reduction in human colorectal carcinoma and with ageing. Nature 346,866-868. Heacock, M . , Spangler, E., Riha, K., Puizina, J., and Shippen, D. E. (2004). Molecular analysis of telomere fusions in Arabidopsis: multiple pathways for chromosome end-joining. E M B O J 23, 2304-2313. Hemann, M . T., Strong, M . A . , Hao, L. Y . , and Greider, C. W. (2001). The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell 107, 67-77. Henderson, E. R., and Blackburn, E. H . (1989). A n overhanging 3' terminus is a conserved feature of telomeres. Mol Cell Biol 9, 345-348. Henson, J. D., Neumann, A . A . , Yeager, T. R., and Reddel, R. R. (2002). Alternative lengthening of telomeres in mammalian cells. Oncogene 21, 598-610. Herrera, E., Samper, E., Martin-Caballero, J., Flores, J. M . , Lee, H . W., and Blasco, M . A . (1999). Disease states associated with telomerase deficiency appear earlier in mice with short telomeres. E M B O J 18, 2950-2960. Hill, A . A . , Hunter, C. P., Tsung, B. T., Tucker-Kellogg, G., and Brown, E. L . (2000). Genomic analysis of gene expression in C. elegans. Science 290, 809-812. Hirst, M . C , and White, P. J. (1998). Cloned human FMR1 trinucleotide repeats exhibit a length- and orientation-dependent instability suggestive of in vivo lagging strand secondary structure. Nucleic Acids Res 26, 2353-2358. Hisama, F. M . , Chen, Y . H., Meyn, M . S., Oshima, J., and Weissman, S. M . (2000). W R N or telomerase constructs reverse 4-nitroquinoline 1-oxide sensitivity in transformed Werner syndrome fibroblasts. Cancer Res 60,2372-2376. Hodgkin, J., and Doniach, T. (1997). Natural variation and copulatory plug formation in Caenorhabditis elegans. Genetics 146,149-164. Hoeijmakers, J. H . (2001). Genome maintenance mechanisms for preventing cancer. Nature 411, 366-374. Holstege, F. C , van der Vliet, P. C , and Timmers, H . T. (1996). Opening of an R N A polymerase II promoter occurs in two distinct steps and requires the basal transcription factors HE and IIH. E M B O J 15, 1666-1677.  129  Holt, M . R., and Koffer, A . (2001). Cell motility: proline-rich proteins promote protrusions. Trends Cell Biol 11, 38-46. Houghtaling, B . R., Cuttonaro, L., Chang, W., and Smith, S. (2004). A dynamic molecular link between the telomere length regulator TRF1 and the chromosome end protector TRF2. Curr Biol 14, 1621-1631. Huang, S., L i , B., Gray, M . D., Oshima, J., Mian, I. S., and Campisi, J. (1998). The premature ageing syndrome protein, W R N , is a 3'~>5' exonuclease. Nat Genet 20, 114-116. Huffman, K . E., Levene, S. D., Tesmer, V . M . , Shay, J. W., and Wright, W. E. (2000). Telomere shortening is proportional to the size of the G-rich telomeric 3'-overhang. J Biol Chem 275, 19719-19722. Hyrien, O., Marie, C , and Mechali, M . (1995). Transition in specification of embryonic metazoan D N A replication origins. Science 270, 994-997. Ionov, Y . , Peinado, M . A., Malkhosyan, S., Shibata, D., and Perucho, M . (1993). Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363, 558-561. Jacob, N . K., Kirk, K. E., and Price, C. M . (2003). Generation of telomeric G strand overhangs involves both G and C strand cleavage. Mol Cell 11, 1021-1032. Jacob, N . K., Skopp, R., and Price, C. M . (2001). G-overhang dynamics at Tetrahymena telomeres. E M B O J 20, 4299-4308. Joeng, K . S., Song, E. J., Lee, K. J., and Lee, J. (2004). Long lifespan in worms with long telomeric D N A . Nat Genet 36, 607-611. Johnson, F. B., Marciniak, R. A., McVey, M . , Stewart, S. A., Hahn, W. C , and Guarente, L . (2001). The Saccharomyces cerevisiae W R N homolog Sgslp participates in telomere maintenance in cells lacking telomerase. E M B O J 20, 905-913. Johnston, B. H . (1988). The SI-sensitive form of d(C-T)n.d(A-G)n: chemical evidence for a three-stranded structure in plasmids. Science 241,1800-1804. Jorgensen, E. M . , and Mango, S. E. (2002). The art and design of genetic screens: caenorhabditis elegans. Nat Rev Genet 3, 356-369. Kamath, R. S., Fraser, A . G., Dong, Y . , Poulin, G., Durbin, R., Gotta, M . , Kanapin, A., Le Bot, N . , Moreno, S., Sohrmann, M . , et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using R N A i . Nature 421, 231-237.  130  Kamath, R. S., Martinez-Campos, M . , Zipperlen, P., Fraser, A . G., and Ahringer, J. (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded R N A in Caenorhabditis elegans. Genome Biol 2, RESEARCH0002. Kang, C , Zhang, X . , Ratliff, R., Moyzis, R., and Rich, A . (1992). Crystal structure of fourstranded Oxytricha telomeric D N A . Nature 356,126-131. Kang, S., Jaworski, A., Ohshima, K., and Wells, R. D. (1995a). Expansion and deletion of C T G repeats from human disease genes are determined by the direction of replication in E. coli. Nat Genet 10, 213-218. Kang, S., Ohshima, K., Shimizu, M . , Amirhaeri, S., and Wells, R. D. (1995b). Pausing of D N A synthesis in vitro at specific loci in C T G and C G G triplet repeats from human hereditary disease genes. J Biol Chem 270, 27014-27021. Karlseder, J., Smogorzewska, A., and de Lange, T. (2002). Senescence induced by altered telomere state, not telomere loss. Science 295,2446-2449. Karow, J. K., Chakraverty, R. K., and Hickson, I. D. (1997). The Bloom's syndrome gene product is a 3-5' D N A helicase. J Biol Chem 272, 30611-30614. Karow, J. K., Constantinou, A., L i , J. L., West, S. C , and Hickson, I. D. (2000). The Bloom's syndrome gene product promotes branch migration of holliday junctions. Proc Natl Acad Sci U S A 97, 6504-6508. Kashi, Y . , King, D., and Soller, M . (1997). Simple sequence repeats as a source of quantitative genetic variation. Trends Genet 13, 74-78. Kastan, M . B., and Bartek, J. (2004). Cell-cycle checkpoints and cancer. Nature 432, 316323. Keightley, P. D., and Charlesworth, B . (2005). Genetic instability of C. elegans comes naturally. Trends Genet 21, 67-70. Khakhar, R. R., Cobb, J. A., Bjergbaek, L., Hickson, I. D., and Gasser, S. M . (2003). RecQ helicases: multiple roles in genome maintenance. Trends Cell Biol 13, 493-501. K i m , S. H., Beausejour, C , Davalos, A . R., Kaminker, P., Heo, S. J., and Campisi, J. (2004). TIN2 mediates functions of TRF2 at human telomeres. J Biol Chem 279,43799-43804. Kim, S. H., Kaminker, P., and Campisi, J. (1999). TIN2, a new regulator of telomere length in human cells. Nat Genet 23,405-412.  131  Kipling, D., and Cooke, H. J. (1990). Hypervariable ultra-long telomeres in mice. Nature 347, 400-402. Kitao, S., Ohsugi, I., Ichikawa, K., Goto, M . , Furuichi, Y . , and Shimamoto, A . (1998). Cloning of two new human helicase genes of the RecQ family: biological significance of multiple species in higher eukaryotes. Genomics 54, 443-452. Klobutcher, L. A., Swanton, M . T., Donini, P., and Prescott, D. M . (1981). A l l gene-sized D N A molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus. Proc Natl Acad Sci U S A 78, 3015-3019. Kolodner, R. D., and Marsischky, G. T. (1999). Eukaryotic D N A mismatch repair. Curr Opin Genet Dev 9, 89-96. Kolquist, K . A., Ellisen, L. W., Counter, C. M . , Meyerson, M . , Tan, L. K., Weinberg, R. A., Haber, D. A., and Gerald, W. L . (1998). Expression of TERT in early premaiignant lesions and a subset of cells in normal tissues. Nat Genet 19,182-186. Korolev, S., Hsieh, J., Gauss, G. H., Lohman, T. M . , and Waksman, G. (1997). Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded D N A and A D P . Cell 90, 635-647. Kyrion, G., Boakye, K . A., and Lustig, A . J. (1992). C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae. Mol Cell Biol 72,5159-5173. Lahue, R. S., A u , K . G., and Modrich, P. (1989). D N A mismatch correction in a defined system. Science 245, 160-164. Lai, C. K., Mitchell, J. R., and Collins, K . (2001). R N A binding domain of telomerase reverse transcriptase. M o l Cell Biol 21, 990-1000. Lansdorp, P. M . , Verwoerd, N . P., van de Rijke, F. M . , Dragowska, V., Little, M . T., Dirks, R. W., Raap, A . K., and Tanke, H . J. (1996). Heterogeneity in telomere length of human chromosomes. Hum M o l Genet 5, 685-691. Lapidot, A., Baran, N . , and Manor, H . (1989). (dT-dC)n and (dG-dA)n tracts arrest single stranded D N A replication in vitro. Nucleic Acids Res 17, 883-900. Laroche, T., Martin, S. G., Gotta, M . , Gorham, H . C , Pryde, F. E., Louis, E. J., and Gasser, S. M . (1998). Mutation of yeast K u genes disrupts the subnuclear organization of telomeres. Curr Biol 8, 653-656.  132  Lebel, M . , Spillare, E. A., Harris, C. C , and Leder, P. (1999). The Werner syndrome gene product co-purifies with the D N A replication complex and interacts with P C N A and topoisomerase I. J Biol Chem 274, 37795-37799. LeBowitz, J. H., and McMacken, R. (1986). The Escherichia coli dnaB replication protein is a D N A helicase. J Biol Chem 261, 4738-4748. Lee, H . W., Blasco, M . A., Gottlieb, G. J., Horner, J. W., 2nd, Greider, C. W., and DePinho, R. A . (1998). Essential role of mouse telomerase in highly proliferative organs. Nature 392, 569-574. Leeds, P., Peltz, S. W., Jacobson, A., and Culbertson, M . R. (1991). The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev 5,2303-2314. Lehmann, A . R. (2001). The xeroderma pigmentosum group D (XPD) gene: one gene, two functions, three diseases. Genes Dev 15,15-23. Lei, M . , Baumann, P., and Cech, T. R. (2002). Cooperative binding of single-stranded telomeric D N A by the Potl protein of Schizosaccharomyces pombe. Biochemistry 41, 14560-14568. Lei, M . , Podell, E. R., and Cech, T. R. (2004). Structure of human POT1 bound to telomeric single-stranded D N A provides a model for chromosome end-protection. Nat Struct Mol Biol 11, 1223-1229. Lendvay, T. S., Morris, D. K., Sah, J., Balasubramanian, B., and Lundblad, V . (1996). Senescence mutants of Saccharomyces cerevisiae with a defect in telomere replication identify three additional EST genes. Genetics 144, 1399-1412. Levinson, G., and Gutman, G. A . (1987). Slipped-strand mispairing: a major mechanism for D N A sequence evolution. M o l Biol Evol 4,203-221. Levy, D. L., and Blackburn, E. H. (2004). Counting of Riflp and Rif2p on Saccharomyces cerevisiae telomeres regulates telomere length. M o l Cell Biol 24,10857-10867. Lew, A., Rutter, W. J., and Kennedy, G. C. (2000). Unusual D N A structure of the diabetes susceptibility locus IDDM2 and its effect on transcription by the insulin promoter factor Pur1/MAZ. Proc Natl Acad Sci U S A 97, 12508-12512. L i , B., and Lustig, A . J. (1996). A novel mechanism for telomere size control in Saccharomyces cerevisiae. Genes Dev 10, 1310-1326.  133  L i , G. Z., Eller, M . S., Firoozabadi, R., and Gilchrest, B. A. (2003). Evidence that exposure of the telomere 3' overhang sequence induces senescence. Proc Natl Acad Sci U S A 100, 527-531. Lillard-Wetherell, K., Machwe, A., Langland, G. T., Combs, K . A., Behbehani, G. K., Schonberg, S. A., German, J., Turchi, J. J., Orren, D. K., and Groden, J. (2004). Association and regulation of the B L M helicase by the telomere proteins TRF1 and TRF2. Hum M o l Genet 13,1919-1932. Lim, C. S., Mian, I. S., Dernburg, A . F., and Campisi, J. (2001). C. elegans clk-2, a gene that limits life span, encodes a telomere length regulator similar to yeast telomere binding protein Tel2p. Curr Biol 11, 1706-1710. Lin, J., Ly, H., Hussain, A., Abraham, M . , Pearl, S., Tzfati, Y . , Parslow, T. G., and Blackburn, E. H . (2004). A universal telomerase R N A core structure includes structured motifs required for binding the telomerase reverse transcriptase protein. Proc Natl Acad Sci U S A 101, 14713-14718. Lin, J. J., and Zakian, V . A . (1996). The Saccharomyces CDC13 protein is a single-strand TGI-3 telomeric DNA-binding protein in vitro that affects telomere behavior in vivo. Proc Natl Acad Sci U S A 93, 13760-13765. Lindor, N . M . , Furuichi, Y . , Kitao, S., Shimamoto, A., Arndt, C , and Jalal, S. (2000). Rothmund-Thomson syndrome due to RECQ4 helicase mutations: report and clinical and molecular comparisons with Bloom syndrome and Werner syndrome. A m J Med Genet 90, 223-228. Lingner, J., Cech, T. R., Hughes, T. R., and Lundblad, V . (1997a). Three Ever Shorter Telomere (EST) genes are dispensable for in vitro yeast telomerase activity. Proc Natl Acad Sci U S A 94, 11190-11195. Lingner, J., Hendrick, L. L., and Cech, T. R. (1994). Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. Genes Dev 8, 1984-1998. Lingner, J., Hughes, T. R., Shevchenko, A., Mann, M . , Lundblad, V., and Cech, T. R. (1997b). Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 276, 561-567. Liu, B., Nicolaides, N . C , Markowitz, S., Willson, J. K., Parsons, R. E., Jen, J., Papadopolous, N . , Peltomaki, P., de la Chapelle, A., Hamilton, S. R., and et al. (1995). Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability. Nat Genet 9,48-55.  134  Liu, B., Parsons, R., Papadopoulos, N . , Nicolaides, N . C , Lynch, H . T., Watson, P., Jass, J. R., Dunlop, M . , Wyllie, A., Peltomaki, P., et al. (1996). Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nat Med 2,169-174. Liu, D., O'Connor, M . S., Qin, J., and Songyang, Z. (2004a). Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins. J Biol Chem 279, 51338-51342. Liu, D., Safari, A., O'Connor, M . S., Chan, D. W., Laegeler, A., Qin, J., and Songyang, Z. (2004b). PTOP interacts with POT1 and regulates its localization to telomeres. Nat Cell Biol 6, 673-680. Liu, K., Schoonmaker, M . M . , Levine, B. L., June, C. H., Hodes, R. J., and Weng, N . P. (1999). Constitutive and regulated expression of telomerase reverse transcriptase (hTERT) in human lymphocytes. Proc Natl Acad Sci U S A 96, 5147-5152. Liu, Y . , Kha, H., Ungrin, M . , Robinson, M . O., and Harrington, L. (2002). Preferential maintenance of critically short telomeres in mammalian cells heterozygous for mTert. Proc Natl Acad Sci U S A 99, 3597-3602. Liu, Y . , Snow, B . E., Hande, M . P., Yeung, D., Erdmann, N . J., Wakeham, A., Itie, A . , Siderovski, D. P., Lansdorp, P. M . , Robinson, M . O., and Harrington, L. (2000). The telomerase reverse transcriptase is limiting and necessary for telomerase function in vivo. C u r r B i o l i O , 1459-1462. Loayza, D., and De Lange, T. (2003). POT1 as a terminal transducer of TRF1 telomere length control. Nature 423, 1013-1018. Lue, N . F., Buchman, A . R., and Kornberg, R. D. (1989). Activation of yeast R N A polymerase II transcription by a thymidine-rich upstream element in vitro. Proc Natl Acad Sci U S A 86, 486-490. Lundblad, V., and Blackburn, E. H . (1993). A n alternative pathway for yeast telomere maintenance rescues estl- senescence. Cell 73, 347-360. Lundblad, V., and Szostak, J. W. (1989). A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 57, 633-643. Lustig, A . J. (2001). Cdcl3 subcomplexes regulate multiple telomere functions. Nat Struct Biol 8, 297-299. Lustig, A . J. (2003). Clues to catastrophic telomere loss in mammals from yeast telomere rapid deletion. Nat Rev Genet 4, 916-923.  135  Lustig, A . J., Kurtz, S., and Shore, D. (1990). Involvement of the silencer and U A S binding protein RAP1 in regulation of telomere length. Science 250, 549-553. MacNeill, S. A . (2001). D N A replication: partners in the Okazaki two-step. Curr Biol 11, R842-844. Makarov, V . L., Hirose, Y . , and Langmore, J. P. (1997). Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell 88, 657-666. Malik, H . S., Burke, W. D., and Eickbush, T. H . (2000). Putative telomerase catalytic subunits from Giardia lamblia and Caenorhabditis elegans. Gene 251, 101-108. Marcand, S., Gilson, E., and Shore, D. (1997). A protein-counting mechanism for telomere length regulation in yeast. Science 275, 986-990. Martens, U . M . , Zijlmans, J. M . , Poon, S. S., Dragowska, W., Y u i , J., Chavez, E. A., Ward, R. K., and Lansdorp, P. M . (1998). Short telomeres on human chromosome 17p. Nat Genet 18, 76-80. Masutomi, K., Y u , E. Y . , Khurts, S., Ben-Porath, I., Currier, J. L., Metz, G. B., Brooks, M . W., Kaneko, S., Murakami, S., DeCaprio, J. A., et al. (2003). Telomerase maintains telomere structure in normal human cells. Cell 114, 241-253. Maurer, D. J., O'Callaghan, B. L., and Livingston, D. M . (1996). Orientation dependence of trinucleotide C A G repeat instability in Saccharomyces cerevisiae. M o l Cell Biol 16, 66176622. McEachern, M . J., and Blackburn, E. H. (1994). A conserved sequence motif within the exceptionally diverse telomeric sequences of budding yeasts. Proc Natl Acad Sci U S A 91, 3453-3457. McElligott, R., and Wellinger, R. J. (1997). The terminal D N A structure of mammalian chromosomes. E M B O J 16, 3705-3714. McGlynn, P., and Lloyd, R. G. (2002). Recombinational repair and restart of damaged replication forks. Nat Rev M o l Cell Biol 3, 859-870. Meloni, R., Albanese, V., Ravassard, P., Treilhou, F., and Mallet, J. (1998). A tetranucleotide polymorphic microsatellite, located in the first intron of the tyrosine hydroxylase gene, acts as a transcription regulatory element in vitro. Hum Mol Genet 7, 423-428.  136  Menichini, P., and Linial, M . (2001). S U V i and B A C H 1 : a new subfamily of mammalian helicases? Mutat Res 487, 67-71. Metzgar, D., Bytof, J., and Wills, C. (2000). Selection against frameshift mutations limits microsatellite expansion in coding D N A . Genome Res 10, 72-80. Miret, J. J., Pessoa-Brandao, L., and Lahue, R. S. (1997). Instability of C A G and C T G trinucleotide repeats in Saccharomyces cerevisiae. M o l Cell Biol 17, 3382-3387. Mitas, M . (1997). Trinucleotide repeats associated with human disease. Nucleic Acids Res 25, 2245-2254. Mohaghegh, P., Karow, J. K., Brosh Jr, R. M . , Jr., Bohr, V . A., and Hickson, I. D. (2001). The Bloom's and Werner's syndrome proteins are D N A structure-specific helicases. Nucleic Acids Res 29, 2843-2849. Morin, G. B . (1989). The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes T T A G G G repeats. Cell 59, 521-529. Munoz-Jordan, J. L., Cross, G. A., de Lange, T., and Griffith, J. D. (2001). t-loops at trypanosome telomeres. E M B O J 20, 579-588. Murti, K . G., and Prescott, D. M . (1999). Telomeres of polytene chromosomes in a ciliated protozoan terminate in duplex D N A loops. Proc Natl Acad Sci U S A 96, 14436-14439. Nakamura, T. M . , Cooper, J. P., and Cech, T. R. (1998). Two modes of survival of fission yeast without telomerase. Science 282,493-496. Nakamura, T. M . , Morin, G. B., Chapman, K . B., Weinrich, S. L., Andrews, W. H., Lingner, J., Harley, C. B., and Cech, T. R. (1997). Telomerase catalytic subunit homologs from fission yeast and human. Science 277, 955-959. Nelson, H . C , Finch, J. T., Luisi, B. F., and Klug, A . (1987). The structure of an oligo(dA).oligo(dT) tract and its biological implications. Nature 330, 221-226. Nugent, C. I., Hughes, T. R., Lue, N . F., and Lundblad, V . (1996). Cdcl3p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance. Science 274, 249-252. Nugent, C. I., and Lundblad, V . (1998). The telomerase reverse transcriptase: components and regulation. Genes Dev 12,1073-1085.  137  Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D. (1996). Cloning, characterization, and properties of seven triplet repeat D N A sequences. J Biol Chem 271, 16773-16783. Opresko, P. L., Otterlei, M . , Graakjaer, J., Bruheim, P., Dawut, L., Kolvraa, S., May, A . , Seidman, M . M . , and Bohr, V . A . (2004). The Werner Syndrome Helicase and Exonuclease Cooperate to Resolve Telomeric D Loops in a Manner Regulated by TRF1 and TRF2. M o l Cell 14,161-114. Opresko, P. L., von Kobbe, C , Laine, J. P., Harrigan, J., Hickson, I. D., and Bohr, V . A . (2002). Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases. J Biol Chem 277, 41110-41119. Ouyang, H., Shiwaku, H . O., Hagiwara, H., Miura, K., Abe, T., Kato, Y . , Ohtani, H., Shiiba, K., Souza, R. F., Meltzer, S. J., and Horii, A . (1997). The insulin-like growth factor II receptor gene is mutated in genetically unstable cancers of the endometrium, stomach, and colorectum. Cancer Res 57, 1851-1854. Panyutin, I. G., Kovalsky, O. I., Budowsky, E. I., Dickerson, R. E., Rikhirev, M . E., and Lipanov, A . A . (1990). G-DNA: a twice-folded D N A structure adopted by single-stranded oligo(dG) and its implications for telomeres. Proc Natl Acad Sci U S A 87, 867-870. Park, E., Guzder, S. N . , Koken, M . H., Jaspers-Dekker, I., Weeda, G., Hoeijmakers, J. H., Prakash, S., and Prakash, L. (1992). RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B D N A repair gene, is essential for viability. Proc Natl Acad Sci U S A 89, 11416-11420. Parkinson, G. N . , Lee, M . P., and Neidle, S. (2002). Crystal structure of parallel quadruplexes from human telomeric D N A . Nature 417, 876-880. Pause, A., and Sonenberg, N . (1992). Mutational analysis of a D E A D box R N A helicase: the mammalian translation initiation factor eIF-4A. E M B O J 11, 2643-2654. Pelengaris, S., Rudolph, B., and Littlewood, T. (2000). Action of Myc in vivo - proliferation and apoptosis. Curr Opin Genet Dev 10, 100-105. Peltomaki, P. (2001). D N A mismatch repair and cancer. Mutat Res 488, 77-85. Peng, Y . , Mian, I. S., and Lue, N . F. (2001). Analysis of telomerase processivity: mechanistic similarity to HIV-1 reverse transcriptase and role in telomere maintenance. M o l Cell 7, 12011211. Pennock, E., Buckley, K., and Lundblad, V . (2001). Cdcl3 delivers separate complexes to the telomere for end protection and replication. Cell 104, 387-396.  138  Petes, T. D., Greenwell, P. W., and Dominska, M . (1997). Stabilization of microsatellite sequences by variant repeats in the yeast Saccharomyces cerevisiae. Genetics 146,491-498. Phan, A . T., Modi, Y . S., and Patel, D. J. (2004). Propeller-type parallel-stranded G quadruplexes in the human c-myc promoter. J A m Chem Soc 126, 8710-8716. Poulin, G., Nandakumar, R., and Ahringer, J. (2004). Genome-wide R N A i screens in Caenorhabditis elegans: impact on cancer research. Oncogene 23, 8340-8345. Price, C. M . , and Cech, T. R. (1987). Telomeric DNA-protein interactions of Oxytricha macronuclear D N A . Genes Dev / , 783-793. Prolla, T. A . , Christie, D. M . , and Liskay, R. M . (1994). Dual requirement in yeast D N A mismatch repair for M L H 1 and PMS1, two homologs of the bacterial mutL gene. M o l Cell Biol 14, 407-415. Prowse, K . R., Avilion, A . A., and Greider, C. W. (1993). Identification of a nonprocessive telomerase activity from mouse cells. Proc Natl Acad Sci U S A 90, 1493-1497. Rampino, N . , Yamamoto, H., Ionov, Y . , L i , Y . , Sawai, H., Reed, J. C , and Perucho, M . (1997). Somatic frameshift mutations in the B A X gene in colon cancers of the microsatellite mutator phenotype. Science 275, 967-969. Richard, G . F., and Paques, F. (2000). Mini- and microsatellite expansions: the recombination connection. E M B O Rep 1, 122-126. Richardson, C. C. (1983). Bacteriophage T7: minimal requirements for the replication of a duplex D N A molecule. Cell 33, 315-317. Riha, K., McKnight, T. D., Fajkus, J., Vyskot, B., and Shippen, D. E. (2000). Analysis of the G-overhang structures on plant telomeres: evidence for two distinct telomere architectures. Plant J 23, 633-641. Riha, K., McKnight, T. D., Griffmg, L. R., and Shippen, D. E. (2001). Living with genome instability: plant responses to telomere dysfunction. Science 291,1797-1800. Ripley, L. S. (1990). Frameshift mutation: determinants of specificity. Annu Rev Genet 24, 189-213. Rodriguez, M . , Y u , X . , Chen, J., and Songyang, Z. (2003). Phosphopeptide binding specificities of BRCA1 COOH-terminal (BRCT) domains. J Biol Chem 278, 52914-52918.  139  Rolfsmeier, M . L., Dixon, M . J., Pessoa-Brandao, L., Pelletier, R., Miret, J. J., and Lahue, R. S. (2001). Cis-elements governing trinucleotide repeat instability in Saccharomyces cerevisiae. Genetics 157, 1569-1579. Romero, D. P., and Blackburn, E. H . (1991). A conserved secondary structure for telomerase R N A . Cell 67, 343-353. Rosenbluth, R. E., and Baillie, D. L . (1981). The genetic analysis of a reciprocal translocation, eTl(III; V ) , in Caenorhabditis elegans. Genetics 99, 415-428. Rosenbluth, R. E., Cuddeford, C , and Baillie, D. L . (1983). Mutagenesis in Caenorhabditis elegans : I. A rapid eukaryotic mutagen test system using the reciprocal translocation, eTI(III;V). Mutat Res 110, 39-48. Rosenbluth, R. E., Johnsen, R. C , and Baillie, D. L . (1990). Pairing for recombination in L G V of Caenorhabditis elegans: a model based on recombination in deficiency heterozygotes. Genetics 124, 615-625. Rudolph, K . L., Chang, S., Lee, H . W., Blasco, M . , Gottlieb, G. J., Greider, C , and DePinho, R. A . (1999). Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell 96, 701-712. Rufer, N . , Dragowska, W., Thornbury, G., Roosnek, E., and Lansdorp, P. M . (1998). Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol 16,1A3>-1A1. Runge, K . W., and Zakian, V . A . (1996). TEL2, an essential gene required for telomere length regulation and telomere position effect in Saccharomyces cerevisiae. M o l Cell Biol 76,3094-3105. Sakamoto, N . , Chastain, P. D., Parniewski, P., Ohshima, K., Pandolfo, M . , Griffith, J. D., and Wells, R. D. (1999). Sticky D N A : self-association properties of long G A A . T T C repeats in R . R . Y triplex structures from Friedreich's ataxia. M o l Cell 3, 465-475. Samadashwily, G. M . , Raca, G., and Mirkin, S. M . (1997). Trinucleotide repeats affect D N A replication in vivo. Nat Genet 17, 298-304. Sancar, A . , Lindsey-Boltz, L. A . , Unsal-Kacmaz, K., and Linn, S. (2004). Molecular mechanisms of mammalian D N A repair and the D N A damage checkpoints. Annu Rev Biochem 73, 39-85.  140  Sasaki, T., Sawado, T., Yamaguchi, M . , and Shinomiya, T. (1999). Specification of regions of D N A replication initiation during embryogenesis in the 65-kilobase DNApolalpha-dE2F locus of Drosophila melanogaster. Mol Cell Biol 19, 547-555. Schaeffer, L., Moncollin, V., Roy, R., Staub, A., Mezzina, M . , Sarasin, A., Weeda, G., Hoeijmakers, J. H., and Egly, J. M . (1994). The ERCC2/DNA repair protein is associated with the class IIBTF2/TFIIH transcription factor. E M B O J 13, 2388-2392. Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J. H . , Chambon, P., and Egly, J. M . (1993). D N A repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science 260, 58-63. Schofield, M . J., and Hsieh, P. (2003). D N A mismatch repair: molecular mechanisms and biological function. Annu Rev Microbiol 57, 579-608. Schorderet, D. F., and Gartler, S. M . (1992). Analysis of CpG suppression in methylated and nonmethylated species. Proc Natl Acad Sci U S A 89, 957-961. Schulz, V . P., Zakian, V . A., Ogburn, C. E., McKay, J., Jarzebowicz, A . A., Edland, S. D., and Martin, G. M . (1996). Accelerated loss of telomeric repeats may not explain accelerated replicative decline of Werner syndrome cells. Hum Genet 97, 750-754. Sen, D., and Gilbert, W. (1988). Formation of parallel four-stranded complexes by guaninerich motifs in D N A and its implications for meiosis. Nature 334, 364-366. Shafer, R. H., and Smirnov, I. (2000). Biological aspects of D N A / R N A quadruplexes. Biopolymers 56, 209-227. Shampay, J., and Blackburn, E. H . (1988). Generation of telomere-length heterogeneity in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 85, 534-538. Shampay, J., Szostak, J. W., and Blackburn, E. H . (1984). D N A sequences of telomeres maintained in yeast. Nature 310, 154-157. Sharma, S., Sommers, J. A., Wu, L., Bohr, V . A., Hickson, I. D., and Brosh, R. M . , Jr. (2004). Stimulation of flap endonuclease-1 by the Bloom's syndrome protein. J Biol Chem 279, 9847-9856. Shimizu, M . , Gellibolian, R., Oostra, B. A., and Wells, R. D. (1996). Cloning, characterization and properties of plasmids containing C G G triplet repeats from the FMR-1 gene. J M o l Biol 258, 614-626.  141  Shiozaki, E. N . , Gu, L., Yan, N . , and Shi, Y . (2004). Structure of the B R C T repeats of B R C A 1 bound to a BACH1 phosphopeptide: implications for signaling. M o l Cell 14, 405412. Shore, D. (1994). RAP1: a protean regulator in yeast. Trends Genet 10, 408-412. Sia, E. A . , Kokoska, R. J., Dominska, M . , Greenwell, P., and Petes, T. D. (1997). Microsatellite instability in yeast: dependence on repeat unit size and D N A mismatch repair genes. M o l Cell Biol 77, 2851-2858. Siddiqui-Jain, A . , Grand, C. L., Bearss, D. J., and Hurley, L. H . (2002). Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c - M Y C transcription. Proc Natl Acad Sci U S A 99, 11593-11598. Simonsson, T., Pecinka, P., and Kubista, M . (1998). D N A tetraplex formation in the control region of c-myc. Nucleic Acids Res 26,1167-1172. Singer, M . S., and Gottschling, D. E. (1994). T L C 1 : template R N A component of Saccharomyces cerevisiae telomerase. Science 266,404-409. Siroky, J., Zluvova, J., Riha, K., Shippen, D. E., and Vyskot, B. (2003). Rearrangements of ribosomal D N A clusters in late generation telomerase-deficient Arabidopsis. Chromosoma 112, 116-123. Smith, F. W., and Feigon, J. (1992). Quadruplex structure of Oxytricha telomeric D N A oligonucleotides. Nature 356,164-168. Smith, G. P. (1976). Evolution of repeated D N A sequences by unequal crossover. Science 191, 528-535. Smith, S., and de Lange, T. (2000). Tankyrase promotes telomere elongation in human cells. Curr Biol 10,1299-1302. Smith, S., Giriat, I., Schmitt, A . , and de Lange, T. (1998). Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Science 282,1484-1487. Smogorzewska, A . , and De Lange, T. (2004). Regulation of telomerase by telomeric proteins. Annu Rev Biochem 73, 177-208. Smogorzewska, A . , Karlseder, J., Holtgreve-Grez, H., Jauch, A . , and de Lange, T. (2002). D N A ligase IV-dependent N H E J of deprotected mammalian telomeres in G I and G2. Curr Biol 12, 1635-1644.  142  Smogorzewska, A., van Steensel, B., Bianchi, A., Oelmann, S., Schaefer, M . R., Schnapp, G., and de Lange, T. (2000). Control of human telomere length by TRF1 and TRF2. Mol Cell Biol 20, 1659-1668. Snapper, C. M . , Marcu, K. B., and Zelazowski, P. (1997). The immunoglobulin class switch: beyond "accessibility". Immunity 6, 217-223. Starita, L . M . , and Parvin, J. D. (2003). The multiple nuclear functions of B R C A 1 : transcription, ubiquitination and D N A repair. Curr Opin Cell Biol 15, 345-350. Starling, J. A., Maule, J., Hastie, N . D., and Allshire, R. C. (1990). Extensive telomere repeat arrays in mouse are hypervariable. Nucleic Acids Res 18, 6881-6888. Stavropoulos, D. J., Bradshaw, P. S., L i , X . , Pasic, I., Truong, K., Ikura, M . , Ungrin, M . , and Meyn, M . S. (2002). The Bloom syndrome helicase B L M interacts with TRF2 in A L T cells and promotes telomeric D N A synthesis. Hum Mol Genet / / , 3135-3144. Stewart, S. A., Ben-Porath, I., Carey, V . J., O'Connor, B . F., Hahn, W. C , and Weinberg, R. A . (2003). Erosion of the telomeric single-strand overhang at replicative senescence. Nat Genet 33, 492-496. Strand, M . , Prolla, T. A., Liskay, R. M . , and Petes, T. D. (1993). Destabilization of tracts of simple repetitive D N A in yeast by mutations affecting D N A mismatch repair. Nature 365, 274-276. Struhl, K . (1985). Naturally occurring poly(dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast. Proc Natl Acad Sci U S A 82, 8419-8423. Subramanya, H . S., Bird, L. E., Brannigan, J. A., and Wigley, D. B . (1996). Crystal structure of a DExx box D N A helicase. Nature 384, 379-383. Sulston, J. (1988). Cell Lineage. In The Nematode Caenorhabditis elegans, W. B . Wood, ed. (Cold Spring Harbor, Cold Spring Harbor Laboratory Press), pp. 123-155. Sulston, J. E., and Horvitz, H . R. (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev Biol 56,110-156. Sulston, J. E., Schierenberg, E., White, J. G., and Thomson, J. N . (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol 100, 64-119. Sun, H., Bennett, R. J., and Maizels, N . (1999). The Saccharomyces cerevisiae Sgsl helicase efficiently unwinds G-G paired DNAs. Nucleic Acids Res 27, 1978-1984.  143  Sun, H., Karow, J. K., Hickson, I. D., and Maizels, N . (1998). The Bloom's syndrome helicase unwinds G4 D N A . J Biol Chem 273, 27587-27592. Sun, H., Yabuki, A., and Maizels, N . (2001). A human nuclease specific for G4 D N A . Proc Natl Acad Sci U S A 98,12444-12449. Sundquist, W. I., and Klug, A . (1989). Telomeric D N A dimerizes by formation of guanine tetrads between hairpin loops. Nature 342, 825-829. Sung, P., Higgins, D., Prakash, L., and Prakash, S. (1988). Mutation of lysine-48 to arginine in the yeast RAD3 protein abolishes its ATPase and D N A helicase activities but not the ability to bind ATP. E M B O J 7, 3263-3269. Taddei, A . , and Gasser, S. M . (2004). Multiple pathways for telomere tethering: functional implications of subnuclear position for heterochromatin formation. Biochim Biophys Acta 1677, 120-128. Taggart, A . K., Teng, S. C , and Zakian, V . A . (2002). Estlp as a cell cycle-regulated activator of telomere-bound telomerase. Science 297, 1023-1026. Taggart, A . K., and Zakian, V . A . (2003). Telomerase: what are the Est proteins doing? Curr Opin Cell Biol 15,275-280. Teixeira, M . T., Arneric, M . , Sperisen, P., and Lingner, J. (2004). Telomere length homeostasis is achieved via a switch between telomerase- extendible and -nonextendible states. Cell 777,323-335. Tijsterman, M . , Pothof, J., and Plasterk, R. H . (2002). Frequent germline mutations and somatic repeat instability in D N A mismatch-repair-deficient Caenorhabditis elegans. Genetics 161, 651-660. Toth, G., Gaspari, Z., and Jurka, J. (2000). Microsatellites in different eukaryotic genomes: survey and analysis. Genome Res 10, 967-981. Trinh, T. Q., and Sinden, R. R. (1991). Preferential D N A secondary structure mutagenesis in the lagging strand of replication in E. coli. Nature 352, 544-547. Tuteja, N . , and Tuteja, R. (2004). Unraveling D N A helicases. Motif, structure, mechanism and function. Eur J Biochem 271,1849-1863. Usdin, K., and Woodford, K. J. (1995). C G G repeats associated with D N A instability and chromosome fragility form structures that block D N A synthesis in vitro. Nucleic Acids Res 23, 4202-4209.  144  van Steensel, B., and de Lange, T. (1997). Control of telomere length by the human telomeric protein TRF1. Nature 385, 740-743. van Steensel, B., Smogorzewska, A . , and de Lange, T. (1998). TRF2 protects human telomeres from end-to-end fusions. Cell 92,401-413. Veldman, T., Etheridge, K . T., and Counter, C. M . (2004). Loss of hPotl function leads to telomere instability and a cut-like phenotype. Curr Biol 14, 2264-2270. Venkitaraman, A . R. (2002). Cancer susceptibility and the functions of B R C A 1 and B R C A 2 . Cell 108, 171-182. Vijayaratnam, V . (2000) Studies of rec-1 function in Caenorhabditis elegans, PhD thesis, University of British Columbia, Vancouver. Wang, R. C , Smogorzewska, A., and de Lange, T. (2004). Homologous recombination generates T-loop-sized deletions at human telomeres. Cell 119, 355-368. Wang, T. F., Kleckner, N . , and Hunter, N . (1999). Functional specificity of MutL homologs in yeast: evidence for three Mlhl-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction. Proc Natl Acad Sci U S A 96, 13914-13919. Wang, W., Seki, M . , Narita, Y . , Sonoda, E., Takeda, S., Yamada, K . , Masuko, T., Katada, T., and Enomoto, T. (2000a). Possible association of B L M in decreasing D N A double strand breaks during D N A replication. E M B O J 19, 3428-3435. Wang, Y . , Cortez, D., Yazdi, P., Neff, N . , Elledge, S. J., and Qin, J. (2000b). B A S C , a super complex of B R C A 1-associated proteins involved in the recognition and repair of aberrant D N A structures. Genes Dev 14, 927-939. Wang, Y . , and Patel, D. J. (1993). Solution structure of the human telomeric repeat d[AG3(T2AG3)3] G-tetraplex. Structure 1, 263-282. Weinrich, S. L., Pruzan, R., Ma, L., Ouellette, M . , Tesmer, V . M . , Holt, S. E., Bodnar, A . G., Lichtsteiner, S., Kim, N . W., Trager, J. B., et al. (1997). Reconstitution of human telomerase with the template R N A component hTR and the catalytic protein subunit hTRT. Nat Genet 77,498-502. Wellinger, R. J., Ethier, K., Labrecque, P., and Zakian, V . A . (1996). Evidence for a new step in telomere maintenance. Cell 85,423-433. Wellinger, R. J., and Sen, D. (1997). The D N A structures at the ends of eukaryotic chromosomes. Eur J Cancer 33, 735-749.  145  Wellinger, R. J., Wolf, A . J., and Zakian, V . A . (1993a). Origin activation and formation of single-strand TGI-3 tails occur sequentially in late S phase on a yeast linear plasmid. M o l Cell Biol 13, 4057-4065. Wellinger, R. J., Wolf, A . J., and Zakian, V . A . (1993b). Saccharomyces telomeres acquire single-strand TGI-3 tails late in S phase. Cell 72, 51-60. Wells, R. D. (1988). Unusual D N A structures. J Biol Chem 263,1095-1098. Wells, R. D. (1996). Molecular basis of genetic instability of triplet repeats. J Biol Chem 271, 2875-2878. White, P. J., Boris, R. H . , and Hirst, M . C. (1999). Stability of the human fragile X (CGG)(n) triplet repeat array in Saccharomyces cerevisiae deficient in aspects of D N A metabolism. M o l Cell Biol 19, 5675-5684. Wicky, C , Villeneuve, A . M . , Lauper, N . , Codourey, L., Tobler, H . , and Muller, F. (1996). Telomeric repeats (TTAGGC)n are sufficient for chromosome capping function in Caenorhabditis elegans. Proc Natl Acad Sci U S A 93, 8983-8988. Wierdl, M . , Dominska, M . , and Petes, T. D. (1997). Microsatellite instability in yeast: dependence on the length of the microsatellite. Genetics 146, 769-779. Williams, M . , and Maizels, N . (1991). LR1, a lipopolysaccharide-responsive factor with binding sites in the immunoglobulin switch regions and heavy-chain enhancer. Genes Dev 5, 2353-2361. Williamson, J. R., Raghuraman, M . K., and Cech, T. R. (1989). Monovalent cation-induced structure of telomeric D N A : the G-quartet model. Cell 59, 871-880. Winkler, G. S., Araujo, S. J., Fiedler, U . , Vermeulen, W., Coin, F., Egly, J. M . , Hoeijmakers, J. H . , Wood, R. D., Timmers, H . T., and Weeda, G. (2000). TFIIH with inactive X P D helicase functions in transcription initiation but is defective in D N A repair. J Biol Chem 275, 4258-4266. Wotton, D., and Shore, D. (1997). A novel Raplp-interacting factor, Rif2p, cooperates with Riflp to regulate telomere length in Saccharomyces cerevisiae. Genes Dev 11, 748-760. Wright, J. H., Gottschling, D. E., and Zakian, V . A . (1992). Saccharomyces telomeres assume a non-nucleosomal chromatin structure. Genes Dev 6, 197-210.  146  Wright, W. E., Tesmer, V . M . , Huffman, K . E., Levene, S. D., and Shay, J. W. (1997). Normal human chromosomes have long G-rich telomeric overhangs at one end. Genes Dev 11, 2801-2809. Wu, L., Davies, S. L., Levitt, N . C., and Hickson, I. D. (2001). Potential role for the B L M helicase in recombinational repair via a conserved interaction with RAD51. J Biol Chem 276, 19375-19381. Wu, L., and Hickson, I. D. (2003). The Bloom's syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870-874. Wyllie, F. S., Jones, C. J., Skinner, J. W., Haughton, M . F., Wallis, C , Wynford-Thomas, D., Faragher, R. G., and Kipling, D. (2000). Telomerase prevents the accelerated cell ageing of Werner syndrome fibroblasts. Nat Genet 24,16-17. Xia, J., Peng, Y . , Mian, I. S., and Lue, N . F. (2000). Identification of functionally important domains in the N-terminal region of telomerase reverse transcriptase. Mol Cell Biol 20, 5196-5207. Xiong, Y . , and Eickbush, T. H. (1990). Origin and evolution of retroelements based upon their reverse transcriptase sequences. E M B O J 9, 3353-3362. Yamada, N . A., Smith, G. A., Castro, A., Roques, C. N . , Boyer, J. C , and Farber, R. A . (2002). Relative rates of insertion and deletion mutations in dinucleotide repeats of various lengths in mismatch repair proficient mouse and mismatch repair deficient human cells. Mutat Res 499,213-225. Yang, Q., Zheng, Y . L., and Harris, C. C. (2005). POT1 and TRF2 cooperate to maintain telomeric integrity. Mol Cell Biol 25,1070-1080. Ye, J. Z., and de Lange, T. (2004). TIN2 is a tankyrase 1 PARP modulator in the TRF1 telomere length control complex. Nat Genet 36, 618-623. Ye, J. Z., Donigian, J. R., van Overbeek, M . , Loayza, D., Luo, Y . , Krutchinsky, A . N.,.Chait, B. T., and de Lange, T. (2004a). TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres. J Biol Chem 279,47264-47271. Ye, J. Z., Hockemeyer, D., Krutchinsky, A . N . , Loayza, D., Hooper, S. M . , Chait, B . T., and De Lange, T. (2004b). POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex. Genes Dev.  147  Y u , G. L., Bradley, J. D., Attardi, L. D., and Blackburn, E. H . (1990). In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature 344, 126-132. Y u , X . , Chini, C. C., He, M , Mer, G., and Chen, J. (2003). The B R C T domain is a phosphoprotein binding domain. Science 302, 639-642. Zalevsky, J., MacQueen, A . J., Duffy, J. B., Kemphues, K. J., and Villeneuve, A . M . (1999). Crossing over during Caenorhabditis elegans meiosis requires a conserved MutS-based pathway that is partially dispensable in budding yeast. Genetics 153,1271-1283. Zhang, L., Y u , J., Willson, J. K., Markowitz, S. D., Kinzler, K . W., and Vogelstein, B . (2001). Short mononucleotide repeat sequence variability in mismatch repair-deficient cancers. Cancer Res 61, 3801-3805. Zhu, L., Hathcock, K . S., Hande, P., Lansdorp, P. M . , Seldin, M . F., and Hodes, R. J. (1998). Telomere length regulation in mice is linked to a novel chromosome locus. Proc Natl Acad Sci U S A 95, 8648-8653. Zijlmans, J. M . , Martens, U . M . , Poon, S. S., Raap, A . K., Tanke, H . J., Ward, R. K., and Lansdorp, P. M . (1997). Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats. Proc Natl Acad Sci U S A 94, 7423-7428.  148  Appendix A . Primers and PCR conditions used in amplification of (G/C)n tracts  Primer pair 390/391 386/387 368/369 400/401 356/357 358/359 152/153 195/196 334/335 338/339 336/337 410/411 412/413 348/349 342/343 398/399 346/347 203/204 396/397 354/355 217/218  Location Y77E11A Y75B7AL Y39A3CR.6 Y41E3 ZC123.3-a ZC123.3-b M03A1.1 M01E10.2 C04C11 Y15E3A F42C5 R03G5 F46H6/C07A12 Y41D4A C18F3 R144 R11B5 F55F3 F49E10 F38A6 B0524.1  Chr IV V III IV I I II III X X IV X X IV IV III X X X V III  Tract G-I4 G-I5  G5TG12 C17  G-I8 C19 C20 C20 C20 C20 C21 C22 C22 G24 G2AG25 C25 C25 C26 G26 C29 C32  149  final [Mg] (mM) 2.4 2 2 2 2 2 2 2 1.6 2 2 2 2 2 2 2 2 2 2 2 2  Ta (°C) 62 62 58 62 58 56 62 60 62 62 62 62 62 62 62 62 62 62 62 62 60  Appendix B. Genomic, spliced and protein sequences ofgklO 2000) >gkl0 genomic  (modified from Vijayaratnam,  sequence  ATGTCCTCAAGCGATGCATTTTGGAGAATGTTCGCAAACAAAAACAAAGG AAAATCGAATACCCGATCGGCATTTCAAGTTGTGAAAGAGGAGCAGCCGT CCACATCAACAGAACCCGATGATAAAAAGCCGTTACACCATGAAATTGCC GGGGAGATGATTAAAAATCCGGCAAAAGGAAAACGAAAGCGGGTTCAGAT CAAAAACGACGAGTATGAGCAGCTCATGATGCTCGGTGTACCGGTACGCG TGCCACGTGGATTGAGCCTCTACTCGACTCAAAAACTAATGATCGTGAGA ATTCTGACGGCGCTGAAAAATAGTCAAAATGTGCTTGGCGAGTCGCCGAC GGGAAGTGGCAAAACTATGGCTCTGCTGGCATCTACGTGTGCATGGCTCA AGCAGTATATCGATGAGAAGCGAGAATCAAAGGAAAAATGTGAAATCCAT GGATTTTCAGGAAAAACACAGGTGGTGTTCACTAACGATATTGGAATACC GCTAGAATCTAAAGTATATGAAGAGCCACCGGAAGAAGAGGAATTCATTG AGCCTGTGCCAGTGAAAAACGATAAACTTTGTAGGATTTTATTTTCAGTG GAGTTTATCATAAATGTTTTTATTTAGTCTATTGAGCCGATAAATCAAAA TGGGCGATTCAATCATACTTTTGACGCAGACGTCTCGATGTCGACTTCAT TTGGAAATCCAAGACCAACAACGACAAGAAGCTCGGCGGGACCCAGAAAC ATGCAGTACAAGGAGGAGAACGCATGGTTTTCAAAGCAATTTTTAAAAAT ATGAAAAAATGTGTTTCAATTTTCAGGCTAGCAGATGCTGCAGCTGACGG AGACGATTGGAAGGATCCGTCAATGTCAGAGACGGGTCATAAACCGATTT CAGAGGGCTGCAAGACTACGATCAGCTTGTGGTGTATGAGTCCAGCATTG GTTAGTACAATATAAGTCCTATAATTCTATAAAACTAATACAAAAATTAA ATTTCAAGCGTGAAAATCGAAATTTCAGTCTTTCTTCGACGCATTCAACG AAACACGTTCTATAGTCCTCGCATCGGGTACTCTGTGCCCAATGGACACT TTGAAAACCGAATTAGGAATGGAATTCAAGCAACAAGTAGAAGGAGATCA AGTCATTAATAAGGATAACATTTTCGCAGCGGTTTTGCCAATTGTAAGGA AAACATTTGGTTAGGTAGCGTCGTGTGGTGCCCTGAGAATTACCGAACGA ATGAAATTTATTTCTTAAAGAGATGACAGGGCACGTGTTGTCAGACAGTC TCATTGCAGGCTGATCTACAAAACATGCGGGAATTTTACCCCAGAAAAAT GTGACGTCAACACACTCTTCATCATGCGAAATCAGTTGAAAAGTCTGCGT CTCTTCTCATTTCTCGAAGATCAAACCAAAATGAGGCAGGAATTAGGATT TAAAATTTTTGATGCAAATTTATGTTAATTTACTTAGCATTAATGCTAGC ATTGAAAAAGTATGTCATTCGTCCTATAAGTCTCCGGCTAGCATACAAGG TTTATTTTCGAAAATACGGTCTAAAAACTACCAAAATAAAAAGGAAAGCC TTATTAATTTAATTGATTTAAAAATTCCAGGGTCCCTTTGGAAATCGCAT TCAGTGCACGTACCGCAACACGTCAGATCCGGAATCTTCATTTTATTGCG AGCTTGGAGCAATTATCAAGTACGTTGGCGGATTATTTTTCGAATTTTCT ACTTTCAGAGAGACTTGAAAAAAATGAATAGATTTTCAACAAAAATGTCA AATTTTGTTTAGGTTATTTAAATCAAGACTTGTTTAAAACAAATATAATT TCTGAAAATTCAAATCGGAAAACTTAGTTTTTGTCTAATTTTTTATAAAA AGTTACAAAAGCCTTGAAAAATGTTCCAATTGTTGGAAAATTTTCGAATG TTAAGAAATCAGCTCATTTTTTGCAGGTATGTATGCAGTAACGTGCCAGC AGGAATTCTATGTTTTCTCCCCAGCTATCGAGTTCTAGATCAATTAAAAC AATGTATGATCCGTAATTCAACAATGCGACAAATCGAAATGAAAAAAGTT GTGCTCTACGAGCCGCGAAGATCCTCAGAATTAACATCAGTAATGGATCA  150  ATTCGATGCGGCGATCTTCGATCCGTCTAGATTCGGTGCAAACATCAACG' GATCTCTTATGTTTGCCGTTTTTCGTGGAAAAGTTTCCGAGGGCATTGAT TTTGCCGATGATCGTGCGCGTGTGGTGATTTCTGTCGGAATTCCGTATCC AAATGCGATGGATGATCAAGTGAATGCGAAAAAGTTGTATAATGATCAGA ATTCCAAGGAAAAGGGGATTTTGACTGGCGATGAGTGGTATACCACGCAG GCCTATCGAGCATTGAATCAGGCTTTGGGAAGGTGAATATATATACAAAG CCGCGTGGAATAAAAACTACGTTTTTTTTATTTAAAAAATGTTATGCTAT GCTACAAATGTTCAAAACACAGCAATTTTCTACTGTAACACATAATTCTG AAATTTGCGCAACATTTTTGACGCACAAAATATCTCGTAGCGAAGACTAC AGTAATCCTTTAAATGACTACCGTAGTGCTTGTGTCGATTTACGGGCTCG ATTTTCGAATTTCCTAATCGAATTTTGACAGCAGTATTTAATTTGTCTTT TTTTTTCTGTATTGTTTTGTCATTTTTGTGTTAAAAAAAAATATTTAAAT GAAAAACGTCCAATATTGCCTTATAGGAAACTCATACAATCGTATTTTTA CATTATTTTCCGTCTATTGCCGGGTTTCACCTGGAAAACCTGCAATAGAC GGGAAATAATGTAAAAATACGTTTGTAAATAGGAGTTTCCTCATCAAAAT GAAGGCAATATTACACGTTTTTTTCTATTAAAACAGTTGGTTAATGAATA AAATTTTTTTAAGTACAAAAATGACTAAACAATACGAACAACGAAGGAAA ATTGAATATCACTGTCCAAATTCGAAAATAAAATCGAGCCCGTAAATCGG CATAAGCGCTACTTTTAAAGAAATACTGTAATTTTCGCTGCGAGATATTT TGCGAGTCAAATATGTTGTAAAATACGCATTCTCAGGATTTAGTGTTCCC GTAATATCAAATCTATAATATCACGTGAAATAAGGGAATCAGTAGAAAAA CCATCTGGCCAAATTTCACTACAATCTAAAATTCATTAAATCTTCAGATG TCTCCGGCACAAAAACGACTGGGGAGCAATGCTAATGATCGATGAACGCC TAGAACGTCAAACTGGAAATCTTGTGGGCGGAGCCTCGTCTGCTCGTGTC TCAAAATGGATTCGTGCTCAACTGAAAAGCTATCCGAGTTTTAAAGAATT TAATGCGAATTTTCGCGAATTCATCCAGCGTCGACACGCTGTTGAGAAAG CAAAAAAAGAGAATTTTTGTGAATGA  >gklO spliced sequence ATGTCCTCAAGCGATGCATTTTGGAGAATGTTCGCAAACAAAAACAAAGG AAAATCGAATACCCGATCGGCATTTCAAGTTGTGAAAGAGGAGCAGCCGT CCACATCAACAGAACCCGATGATAAAAAGCCGTTACACCATGAAATTGCC GGGGAGATGATTAAAAATCCGGCAAAAGGAAAACGAAAGCGGGTTCAGAT CAAAAACGACGAGTATGAGCAGCTCATGATGCTCGGTGTACCGGTACGCG TGCCACGTGGATTGAGCCTCTACTCGACTCAAAAACTAATGATCGTGAGA ATTCTGACGGCGCTGAAAAATAGTCAAAATGTGCTTGGCGAGTCGCCGAC GGGAAGTGGCAAAACTATGGCTCTGCTGGCATCTACGTGTGCATGGCTCA AGCAGTATATCGATGAGAAGCGAGAATCAAAGGAAAAATGTGAAATCCAT GGATTTTCAGGAAAAACACAGGTGGTGTTCACTAACGATATTGGAATACC GCTAGAATCTAAAGTATATGAAGAGCCACCGGAAGAAGAGGAATTCATTG AGCCTGTGCCAGTGAAAAACGATAAACTTTTCTATTGAGCCGATAAATCA AAATGGGCGATTCAATCATACTTTTGACGCAGACGTCTCGATGTCGACTT CATTTGGAAATCCAAGACCAACAACGACAAGAAGCTCGGCGGGACCCAGA AACATGCAGTACAAGGAGGAGAACGCATGGCTAGCAGATGCTGCAGCTGA CGGAGACGATTGGAAGGATCCGTCAATGTCAGAGACGGGTCATAAACCGA TTTCAGAGGGCTGCAAGACTACGATCAGCTTGTGGTGTATGAGTCCAGCA TTGTCTTTCTTCGACGCATTCAACGAAACACGTTCTATAGTCCTCGCATC  151  GGGTACTCTGTGCCCAATGGACACTTTGAAAACCGAATTAGGAATGGAAT TCAAGCAACAAGTAGAAGGAGATCAAGTCATTAATAAGGATAACATTTTC GCAGCGGTTTTGCCAATTGGTCCCTTTGGAAATCGCATTCAGTGCACGTA CCGCAACACGTCAGATCCGGAATCTTCATTTTATTGCGAGCTTGGAGCAA TTATCAAGTATGTATGCAGTAACGTGCCAGCAGGAATTCTATGTTTTCTC CCCAGCTATCGAGTTCTAGATCAATTAAAACAATGTATGATCCGTAATTC AACAATGCGACAAATCGAAATGAAAAAAGTTGTGCTCTACGAGCCGCGAA GATCCTCAGAATTAACATCAGTAATGGATCAATTCGATGCGGCGATCTTC GATCCGTCTAGATTCGGTGCAAACATCAACGGATCTCTTATGTTTGCCGT TTTTCGTGGAAAAGTTTCCGAGGGCATTGATTTTGCCGATGATCGTGCGC GTGTGGTGATTTCTGTCGGAATTCCGTATCCAAATGCGATGGATGATCAA GTGAATGCGAAAAAGTTGTATAATGATCAGAATTCCAAGGAAAAGGGGAT TTTGACTGGCGATGAGTGGTATACCACGCAGGCCTATCGAGCATTGAATC AGGCTTTGGGAAGATGTCTCCGGCACAAAAACGACTGGGGAGCAATGCTA ATGATCGATGAACGCCTAGAACGTCAAACTGGAAATCTTGTGGGCGGAGC CTCGTCTGCTCGTGTCTCAAAATGGATTCGTGCTCAACTGAAAAGCTATC CGAGTTTTAAAGAATTTAATGCGAATTTTCGCGAATTCATCCAGCGTCGA CACGCTGTTGAGAAAGCAAAAAAAGAGAATTTTTGTGAATGA  >translated protein from gklO MSSSDAFWRMFANK^KGKSNTRSAFQVVKEEQPSTSTEPDDKKPLHHEIAGEMIKN PAKG KRKRVQIKNDEYEQLMMLGVPVRVPRGLSLYSTQKLMIVRILTALKNSQNVLGESP TGSG KTMALLASTCAWLKQYIDEKRESKEKCEIHGFSGKTQVVFTNDIGIPLESKVYEEPPE EE EFIEPVPVKNDKLFY-  152  Appendix C. Repeat sequences that are stable in dog-l(gklO)  Primer pair  Location  Chromosome  294/295  C41G7  I  296/297  Y51H4A  IV  Ao  300/301  ZK377  X  A7  306/307  Y43F8B  310/311  C31C9  II  (CT),o  312/313  M01D7  I  (CT),o  318/319  C53H9  I  (GT)  320/321  T13E8  IV  (CA)io  422/423  F12F6  IV  (CAG)  424/425  C06G1  X  (CAG)io  426/427  Y66D12A  III  (CTG)  428/429  C43E11  I  (CTG)io  430/431  F55A8  IV  G(CGG) CG  432/433  Y71G12B  I  (GGC)  436/437  Y48B6A  II  CG(CCG) CC  850/851  F53H1  IV  (CCG)  852/853  ZC123  I  G(CGG) CG  854/855  Y55F3BR  IV  G(CGG) C  856/857  T23F11  III  CG(CGG) G  858/859  Y18D10A  I  (CCG) C  860/861  C18H7  IV  G(GGC)  Tract A  2 9  2  2  (AT)  153  10  10  8  9  6  7  6  7  6  6  6  6  6  Appendix D. Alternation in recombination frequencies in F25H2.13(RNAi)  Recombination frequencies between dpy-11 and unc-42 on chromosome V , and between unc101 and unc-54 on chromosome I, were measured in F25H2.13 R N A i treated N2 as well as in N2 control. Experiments were carried out by feeding N2 males and dpy-11 unc-42 or unc-101 unc-54 hermaphrodites with E. coli expressing the GenePair Fragment F25H2.1 obtained from the C. elegans Chromosome 1 R N A i library (purchased from Julie. Ahringer, U K H G M P Resource Center) for two generations before crossing. Both crossing of parental strains to N2 males and self-crossing in heterozygous F l were also carried out on R N A i plates. The two parental phenotypes and the two recombinant phenotypes were scored in F2 progeny: when dpy-11 unc-42 was used, the parental phenotypes were Wild and Dpy Unc, and the recombinant phenotypes were Dpy and Unc; when unc-101 unc-54 was used, only Wilds (parental phenotype) and Unci01 's (recombinant phenotype) were counted because they have more distinctive phenotypes.  To calculate 95% confidence interval for recombination frequency, the following equations were used (Brenner, 1974): p = l - V(1-2R/T) R = number of recombinants T = total progeny q = 1-p The 95% confidence interval for the number of recombinants is ±1.96Vnpg, where n = number of recombinants. The resulted number is then used to re-calculate the recombination frequency, giving upper and lower 95% confidence intervals.  For the recombination frequency between dpy-11 and unc-42, the number of recombinants was considered to be 2 times Dpy. The number of Unc's was usually smaller than Dpy's, suggesting that Unc-42 had a higher rate of premature death. Total progeny was calculated as the number of Wilds x 4/3 + recombinants, because phenotypic wild-type progeny encompassed mostly + +/+ + and dpy-11 unc-421 + + non-recombinants, and they made up  154  three quarters of total non-recombinants (the other quarter of non-recombinants was the double mutant). In the R N A i treated samples, there were 14 Dpy and 5 Unc recombinants in 805 total progeny. Therefore, p=0.03541 and q=0.96459, and the 95% confidence interval for the number of recombinants was 1.9. Hence, the number of recombinants was 28±1.9, which led to a recombination frequency with 95% confidence interval of 3.2 to 3.7. For the control (fed with regular E. coli), there were 19 Dpy and 13 Unc recombinants out of 1494 progeny. Using similar calculations as above, p = 0.02577, q = 0.9742, and the recombination frequency with 95% confidence interval was 2.4 to 2.7.  For the recombination frequency between unc-101 and unc-54, the number of recombinants was considered to be 2 times Unc-101. Total progeny was again calculated as number of WT x 4/3 + recombinants. For the control, there were 190 recombinants and 1599 total progeny. Therefore, p = 0.1269, q = 0.8731, and the recombination frequency with 95% interval was 11.3 to 12.4. In the R N A i treated samples, there were 162 recombinants in 1106 total progeny. Therefore, p = 0.1591, q = 0.8409, and the recombination frequency with 95% confidence interval was 13.8 to 15.5.  Therefore, in both cases, recombination frequency was increased slightly but significantly in the F25H2.13 R N A i animals. These results suggest that F25H2.13 affects certain aspect of recombination.  155  Appendix E. Telomere length measurement in dog-1(gklO) and a rescued strain by terminal restriction fragment (TRF) analysis. (Figure adapted from the supplementary materials in Cheung et al, 2002)  Telomere length in the indicated generations in dog-1 (gklO) and in a rescued strain (KR3847) was measured by TRF analysis. The construction of the transgenic strain KR3847, in which a wild-type copy of dog-1 was introduced into the mutant background, is described in section 2.2.1.  dog-1 (gk10)  dog-1(gk10) rescued  11 17 25 33 35 45  11 17 25 33 35 45  156  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0092324/manifest

Comment

Related Items