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Identification and characterization of chromosome instability mutants in the yeast Saccharomyces cerevisiae… Yuen, Wing Yee Karen 2007

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IDENTIFICATION AND CHARACTERIZATION OF CHROMOSOME INSTABILITY MUTANTS IN THE YEAST SACCHAROMYCES CEREVISIAE AND IMPLICATIONS TO HUMAN CANCER  by  WING Y E E K A R E N YUEN B.Sc.  (Hon), Simon Fraser University, 2001  A Thesis Submitted in Partial Fulfillment o f the Requirements for the Degree o f D O C T O R OF P H I L O S O P H Y in The Faculty o f Graduate Studies (Medical Genetics)  The University o f British Columbia January 2007  © W i n g Yee Karen Yuen, 2007  A B S T R A C T  Chromosome instability (CLN) is a hallmark o f cancers and may contribute to tumorigenesis. M a n y genes involved i n maintaining chromosome stability are conserved in eukaryotes, and some are mutated i n cancers. The goal o f this thesis is to use Saccharomyces cerevisiae as a model to identify and characterize genes important for chromosome maintenance, investigate the relevance o f C L N to cancer, and develop a strategy to identify candidate therapeutic target genes for selective k i l l i n g o f cancer cells. To systematically identify genes important for chromosome stability, nonessential gene deletion yeast mutants were examined using 3 complementary C I N assays. The chromosome transmission fidelity assay monitors loss o f an artificial chromosome. The bimater assay monitors loss o f heterozygosity at the mating type locus i n homozygous diploid deletion mutants. The a-like faker assay detects loss o f the MAT a mating type locus in haploid deletion mutants. 293 C I N mutants were identified, including genes functioning i n the chromosome or cell cycle, and genes not clearly implicated in chromosome maintenance, such as MMS22, MMS1, RTT101 and RTT107. Phenotypic, genetic and biochemical analyses o f these 4 gene products indicate that they function in double strand break repair. They may form a ubiquitin ligase complex that regulates the level o f some proteins, including Mms22p itself, during D N A damage response. Human homologues o f 10 yeast C I N genes identified were previously shown to be mutated i n cancers, suggesting that other human homologues are candidate cancer genes. 101 human homologues o f yeast C L N genes were sequenced i n a panel o f colorectal cancers, identifying 20 somatic mutations i n 8 genes. In particular, 17 mutations were found in 5 genes involved i n sister chromatid cohesion. Further functional studies should reveal whether mutations in cohesion genes contribute to C I N in cancers. While C I N mutations may contribute to cancer, C I N cancer cells may become inviable when combined with another non-essential mutation, providing the basis for  n  cancer c e l l - s p e c i f i c therapy. M u t a t i o n s i n CTF4, CTF18, a n d DCC1  i n yeast cause  synthetic l e t h a l i t y w h e n c o m b i n e d w i t h mutations i n v a r i o u s C I N genes w h o s e h u m a n h o m o l o g u e s are mutated i n cancers. S u c h analyses i n yeast c a n p r o p o s e p o t e n t i a l d r u g targets i n h u m a n for cancer therapy.  in  TABLE OF CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST OF T A B L E S  ix  LIST OF FIGURES  x  ACKNOWLEDGMENTS  xii  CO-AUTHORSHIP STATEMENT  CHAPTER 1  .  xv  Introduction: Maintenance of Chromosome Stability in  E u k a r y o t e s a n d the R e l a t i o n s h i p w i t h C a n c e r  1.1 Maintenance o f chromosome stability i n eukaryotes 1.1.1 The cell and chromosome cycles i n eukaryotes  1  2 2  1.1.2 Budding yeast as a model organism to study the cell and chromosome cycle.. 4 1.1.3 Biological processes that affect chromosome stability 1.1.3.1 Kinetochores mediate the attachment with mitotic spindles  6 6  1.1.3.2 Mitotic spindle checkpoint  10  1.1.3.3 Sister chromatid cohesion  13  1.2 Chromosome instability and cancer  15  1.2.1 Aneuploidy is a hallmark o f cancer  15  1.2.2 Relationship between a state o f aneuploidy and an increased rate o f chromosome instability (CLN)  17  1.2.3 C I N occurs at early stage o f cancer, and can be a driving force in tumorigenesis  17  1.2.4 Genetic basis o f C I N i n cancer  18  1.2.4.1 Cancer-prone syndromes  18  1.2.4.2 Mutations in mitotic spindle checkpoint  19  1.2.4.3 Misregulation o f kinetochore proteins  22  iv  1.2.4.4 Additional examples o f mutations i n genes involved i n chromosome segregation  24  1.2.4.5 Therapeutic implications  25  1.3 Overview o f thesis  29  CHAPTER 2 Identification of Chromosome Instablity Mutants in the Budding Yeast Saccharomyces cerevisiae and the Implication to Human Cancer  43  2.1 Introduction  44  2.2 Materials and methods  47  2.2.1 Genome-wide screens  47  2.2.1.1 C T F screen  47  2.2.1.2 Bimater screen  48  2.2.1.3 a-like faker screen  49  2.2.2 Strain verification  50  2.2.3 Bioinformatic analysis  51  2.2.3.1 Functional analysis  '.  51  2.2.3.2 B L A S T analysis  52  2.2.3.3 Protein and synthetic lethal interaction network  52  2.2.4 Electrophoretic karyotype o f a-like fakers 2.3 Results  52 53  2.3.1 Genome-wide marker loss screens identify 130 yeast deletion mutants  53  2.3.2 Functional distribution o f yeast C I N genes  56  2.3.3 Integration o f genome-wide phenotypic screen with genetic screens reveals functions o f uncharacterized genes i n chromosome stability maintenance  58  2.3.4 Chromosome loss is the major mechanism o f M 4 r i o s s i n a-like fakers  59  2.3.5 M a n y yeast C I N genes are conserved  60  2.3.6 A strategy for cancer therapy: synthetic lethality and selective cancer cell killing  61  2.4 Discussion  62  v  CHAPTER 3 Identification of Somatic Mutations in Cohesion Genes in Colorectal Cancers with Chromosome Instability  79  3.1 Introduction  80  3.2 Materials and Methods  82  3.2.1 Gene identification  82  3.2.2 Sequencing  82  3.2.3 Yeast SMC1 mutants construction and characterization  82  3.3 Results  84  3.3.1 20 somatic mutations were found in 8 C I N genes  84  3.3.2 Mutation frequency i n comparison to prevalence o f mutations  84  3.3.3 A conserved missense mutation in yeast SMC1 causes mild C L N  85  3.4 Discussion 3.4.1 SMC1L1,  87 CSPG6, NIPBL,  STAG2 and STAG3  87  3.4.2 A L M  87  3A.3RNF20  88  3.4.4 UTX  89  CHAPTER 4 Characterization of MMS22, MMS1, RTT101 and RTT107 in the  Maintenance of Genome Integrity  103  4.1 Introduction  104  4.1.1 RTT107  105  4A.2RTT10P  107  4.1.3 MMS22 mdMMSl  108  4.2 Materials and Methods  :  ".  110  4.2.1 Yeast strains and media  110  4.2.2 Quantification o f chromosome transmission fidelity (ctf)  110  4.2.3 Genome-wide yeast-two-hybrid screens  110  4.2.4 Co-immunoprecipitation  110  vi  4.2.5 Mass spectrometry  111  4.2.6 Survival assay in HO-induced double strand breaks  Ill  4.2.7 Microscopy  Ill  4.3 Results  113  4.3.1 mms22A, mmslA,  rttlOlA  and rttl07A  exhibit chromosome instability  113  4.3.2 mms22A exhibits defects i n cell cycle progression  113  4.3.3 mms22A has reduced survival rate with the introduction o f D S B s  114  4.3.4 Mms22p interacts with replication initiation and D N A repair proteins that may constitute a novel repair pathway  116  4.3.4.1 Mass spectrometry analysis  116  4.3.4.2 Yeast-two-hybrid analysis -  117  4.3.4.3 Genetic interaction analysis  118  4.3.5 R t t l O l p regulates Mms22p  119  4.4 Discussion  122  4.4.1 Conservation of the R t t l O l p complex?  122  4.4.2 Dia2p may play a redundant role with R t t l O l p complex in replication regulation  124  4.4.3 Identifying targets for R t t l O l p ubiquitin ligase  124  CHAPTER 5  Conclusions and Future Directions  151  5.1 Conclusions  152  5.2 Future Directions  154  REFERENCES  156  APPENDICES  183  Appendix 1 H i g h confidence yeast C L N genes identified by the 3 marker loss screens  183  Appendix 2 Lower confidence yeast C I N genes identified by the 3 marker loss screens  195  vii  Appendix 3 Gene ontology (GO) cellular component annotation enrichment among non-essential yeast C L N genes  206  Appendix 4 Gene ontology (GO) biological process annotation enrichment among non-essential yeast C L N genes  217  Appendix 5 Homologues o f budding yeast C I N genes i n human, mouse, worm, fly, and fission yeast  222  Appendix 6 A subset o f yeast C I N genes identify human homologues that are mutated in cancer or are associated with other human diseases  viii  239  LIST O F TABLES  C H A P T E R 1:  Table 1.1 Germline mutations o f C L N and M I N genes causing cancer-prone syndromes  31  Table 1.2 Kinetochore and spindle checkpoint gene mutation or misregulation associated with cancer  33  C H A P T E R 2:  Table 2.1 24 ctf mutants cloned to date  66  Table 2.2 List o f yeast strains used i n chapter II  67  Table 2.3 Human proteins homologous to yeast C I N genes are mutated i n cancer  68  C H A P T E R 3:  Table 3.1 Relationship o f 100 human candidate C I N genes used i n (Wang et al., 2004b) with yeast genes  :  91  Table 3.2 List o f yeast strains used i n chapter III  94  Table 3.3 101 candidate C I N genes analyzed in this study  95  Table 3.4 Somatic mutations identified in candidate C I N genes i n C L N colorectal cancer cells  98  C H A P T E R 4:  Table 4.1 Types D N A lesions generated by various D N A damaging agents  127  Table 4.2 List o f yeast strains used i n chapter I V  128  Table 4.3 Quantification o f chromosome loss ( C L ) , non-disjunction (NDJ) and chromosome gain (CG) by half-sectored assay  ix  131  LIST O F FIGURES  C H A P T E R 1:  Figure 1.1 The budding yeast cell cycle and chromosome cycle  34  Figure 1.2 Organization o f centromere  35  Figure 1.3 The process o f achieving bipolar attachment  36  Figure 1.4 Types o f kinetochore-microtubule attachments  37  Figure 1.5 Structure o f cohesin and a possible mechanism by which it might hold sister chromatids together  38  Figure 1.6 The stages o f mitosis  39  Figure 1.7 Cellular processes involved in replication and segregation o f chromosomes during mitosis  40  Figure 1.8 Multiple roads to aneuploidy  41  Figure 1.9 Flowchart o f therapeutic strategy based on candidate C I N gene identification  42  C H A P T E R 2:  Figure 2.1 Three screen methods  69  Figure 2.2 Three marker loss screens  73  Figure 2.3 130 high confidence nonessential yeast C I N genes  74  Figure 2.4 Functional groups o f 293 C L N genes  75  Figure 2.5 92 protein interactions among 102 C I N proteins  76  Figure 2.6 A - l i k e fakers result from whole chromosome loss, gross chromosomal rearrangement, and gene conversion  77  Figure 2.7. Common synthetic lethal interactions among yeast C I N genes that have human homologs mutated in cancer  78  x  C H A P T E R 3: Figure 3.1 Mutations in human SMC1L1 i n colorectal cancers and analogous mutations in yeast SMC1  99  Figure 3.2 Synthetic lethal interactions o f yeast C I N genes whose human homologs were found mutated in colorectal cancers  102  C H A P T E R 4: Figure 4.1 D N A damage and repair mechanisms  132  Figure 4.2 Two-dimensional hierarchical clustering o f drugs and yeast deletion mutants...J  :  133  Figure 4.3 C e l l cycle and morphology defects of mms22A  137  Figure 4.4 Defects o f mms22A i n double-strand breaks  138  Figure 4.5 Kinetics o f D S B repair  139  Figure 4.6 Mms22p co-immunoprecipitates with R t t l O l p and R t t l 0 7 p  141  Figure 4.7 Physical interaction o f R t t l O l p with M m s l p  142  Figure 4.8 Yeast-two-hybrid interactions using bait protein: (A) Mms22p, (B) R t t l O l p , and (C) Rttl07p  143  Figure 4.9 Physical interactions of Mms22p with M m s l p  144  Figure 4.10 Genetic interactions of mms22A mutants  145  Figure 4.11 Interaction network of MMS22, MMS1, RTT101 and RTT107  146  Figure 4.12 Mms22p expression is regulated by R t t l O l p  148  Figure 4.13 C e l l cycle expression o f R t t l O l p  150  xi  ACKNOWLEDGMENTS The completion o f this thesis would not be possible without the help and support of many people, who I would like to take this opportunity to thank and acknowledge. First, I would like to thank my supervisor Phil Hieter for his guidance and advices throughout the course o f my P h D study. Phil has provided a very supportive lab environment with many great scientists and resourceful colleagues. P h i l also has extensive networking with scientists in the yeast community and worldwide. P h i l has introduced me to many experienced scientists, and he has been instrumental in setting up productive collaborations. I am also thankful for P h i l ' s openness, positivity and generosity. Second, I would like to thank members o f the Hieter lab for a constructive and fun working and learning atmosphere. I want to thank the many helpful post-doctoral fellows/visiting professors in the lab, including V i v i e n Measday, Kristin Batez, Melanie Mayer, Daniel Kornitzer, Shay Ben-Aroya, K i r k M c M a n u s , A m i r Aharoni, and Giora Simchen, who all never hesitated to offer guidance and assistance. I am grateful to grow scientifically with fellow students in the lab. Thanks A n d y Page for introducing me to the basics o f yeast biology and genetics. Thanks Isabelle Pot for teaching me many molecular and biochemical techniques. I would not survive these years without Isabelle's consistent encouragement and our daily energizing 'cookie club'. Thanks B e n Cheng, Elaine L a w , Ben Montpetit, and Jan Stopel for insightful discussion and interactions (and a little bit o f pressure). I am appreciative o f the technical assistance by Teresa K w o k . A n d thanks Irene Barrett and Dave Thomson for keeping the lab i n place. Next, I like to thank collaborators who have contributed to this work. I am indebted to Forrest Spencer at Johns Hopkins University for enormous guidance and support. Her enthusiasm, compassion and understanding always brighten m y days. Thanks also go to Cheryl Warren (from Forrest Spencer's lab) who performed the a-like faker screen and subsequent analysis (Chapter 2); M a r k Flory (from R u d i Aebersold's lab at the Institute o f Systems Biology) who performed the mass spectrometry analysis  xii  (Chapter 4); Tony Hazbun (from Stan Fields' lab at the Yeast Resource Centre) who performed the yeast-two-hybrid analysis (Chapter 4); Shira Goldstein (from Martin Kupiec's lab at Tel A v i v University) who performed the Southern blot and P C R analysis for monitoring the repair kinetics o f double-strand D N A breaks (Chapter 4). T o m Barber, Marcelo Reis (in Victor Velculescu and Bert Vogelstein group at Johns Hopkins University) and Christoph Lengauer have been instrumental to the cancer mutation testing project (Chapter 3). In addition, I need to thank members o f m y advisory committee, Carolyn Brown, A n n Rose, and L i z Conibear, for guidance, directions and support throughout the years, and invaluable feedbacks on m y thesis. I also want to thank M i c h e l Roberge and M i k e Kobor for stimulating discussion and Joanne Fox (in U B C Bioinformatics Centre) for bioinformatic assistance (Chapter 2). Besides, I would like to thank teachers and mentors who have inspired me to pursue a scientific endeavor. M y high school physics teacher i n H o n g K o n g , M r . Y . S . A u , has exposed me to the excitements in doing experiments. Dennis Jewell demonstrated his passion in teaching and supported me tremendously during m y first year in Canada. Working with Jean St. Pierre at Ballard Power Systems i n m y first co-op experience has exposed me to a new perspective i n applied research. M y honor thesis supervisor Lynne Quarmby at Simon Fraser University has demonstrated to me her creativity and enthusiasm in studying cellular biology. Finally, I am thankful to m y parents, M a n d y and John Y u e n , for their unconditional love, continuous trust, support and an unlimited supply o f heart-warming soups! I could not imagine how m y years o f graduate life would be like without the love, back-up, encouragement and understanding o f m y soul mate Simon Chiu. I need to thank my brother K e n , aunt Pamela Cheng, very good friends Rosa Tchao, Helen Leung, Vernice Y u , Ivy N g , Daphne Chow and WaiTak Tsun for their long-distance support as well. I also want to say thanks to the brothers and sisters at Pacific Grace M B Church, especially Oliver Chan and Rev. Issac Chang, for their prayers and support. A n d thank God for all the blessings and challenges.  xm  T h r o u g h o u t this thesis w o r k , I w a s f i n a n c i a l l y supported b y the N a t i o n a l S c i e n c e and E n g i n e e r i n g R e s e a r c h C o u n c i l ( N S E R C ) postgraduate s c h o l a r s h i p s a n d the U n i v e r s i t y o f B r i t i s h C o l u m b i a graduate f e l l o w s h i p , w h o I w o u l d l i k e to thank here.  xiv  CO-AUTHORSHIP STATEMENT Chapter 2 o f this thesis was co-written by Cheryl D . Warren. I designed and performed all the work described i n this chapter except for the following: - a-like faker screen and retest - sequencing o f unique tags o f mutants - electrophoretic karyotyping o f a-like fakers These experiments were performed by Cheryl Warren and O u Chen i n Forrest Spencer's laboratory.  xv  CHAPTER 1 Introduction: Maintenance of Chromosome Stability in Eukaryotes and the Relationship with Cancer  Part o f this chapter has been published. Karen W Y Yuen*, B e n Montpetit* and Phil Hieter ( T h e s e authors contributed equally to this work). (2005) The Kinetochore and Cancer: What's the Connection? Current Opinion in C e l l Biology. 17:1-7.  1  1.1 Maintenance of chromosome stability in eukaryotes 1.1.1 The cell and chromosome cycles in eukaryotes Over 100 years ago, Walter Flemrning first described mitosis (1874), and Theodor Boveri showed the dramatic synchronous separation o f chromosomes during the first mitotic division o f fertilized sea urchin eggs (1902) (reviewed i n (Manchester, 1995; Paweletz, 2001)). The maintenance o f an individual organism requires that each daughter cell receives a full and exact complement o f genetic information from its mother cell. To ensure the conservation o f euploidy (normal number o f chromosomes) i n eukaryotic cells, genetic information must be accurately copied and transmitted to each daughter cell during every mitotic division cycle. Errors i n chromosome segregation (including chromosome non-disjunction and chromosome loss) result in aneuploidy (abnormal number o f chromosomes). Phenotypic consequences o f these imbalances i n chromosome number could be profound and dire. Boveri later postulated that unequal segregation o f chromosomes might be a cause for tumor development and birth defects (1914) (reviewed in (Manchester, 1995)). Indeed, aneuploidy is a hallmark o f cancer, and the relationship between chromosome missegregation and cancer w i l l be discussed i n section 1.2. First, the progression o f a normal mitotic cell cycle is reviewed. The mitotic/somatic cell cycle is divided into 4 phases: G l (growth/gap), S ( D N A synthesis), G 2 and M (mitosis). The M phase is subdivided into: prophase, prometaphase, metaphase, anaphase, and telophase. In prophase o f metazoans, sister chromatids condense, and the nuclear envelope breaks downs. During prometaphase, sister kinetochores undergo the process o f establishing bi-polar orientation with opposite spindle poles. Once bi-polar orientation has been achieved by all kinetochores, the cell enters metaphase when all chromosomes congress to a central position called the metaphase plate. Anaphase then begins and is composed o f 2 steps: anaphase A during which sister chromatids separate, and move away from each other toward spindle poles; and anaphase B when the spindle poles separate by moving in opposite directions. In telophase, chromosomes decondense and a new nuclear envelope forms. Finally, cytokinesis occurs when cytoplasm divides and 2 daughter cells are formed. Each step in  2  the cell cycle has to be executed with high fidelity and coordinated temporally and spatially in order to maintain genetic integrity. C e l l cycle progression is regulated mainly through stage-specific phosphorylation of proteins by cyclin-dependent kinases ( C D K s ) . C D K activity is controlled by both positive and negative regulatory subunits called cyclins, and C D K inhibitors ( C K I ) (e.g. SIC1), respectively. Cyclins are targeted for ubiquitin-mediated degradation at specific stages o f the cell cycle. In addition, key proteins are degraded i n a cell cycle-specific manner to prevent events such as D N A re-replication and centrosome re-duplication. Otherwise, polyploidy (multiple sets o f the normal number o f chromosomes) or aneuploidy could result at an unacceptably high level. Proteins targeted for degradation are first ubiquitylated. Ubiquitin (Ub) is an essential 76-amino acid protein that is conserved i n all eukaryotes. The polyubiquitylation reaction requires enzymes E l - 3 . The ubiquitin-activating enzyme ( E l ) activates U b b y forming unstable thioester bonds with U b . The ubiqui tin-conjugating enzyme (E2) then transfers U b covalently to the substrate. The ubiquitin-ligase (E3) determines the specificity o f the reaction by binding with the substrate and E 2 . One large class o f E3 is cullin-dependent ubiquitin ligase ( C D L ) , which contains a catalytic core that is composed o f a cullin and a R I N G finger protein, and substrate recognition modules. The cullin 'culls' or sorts different substrates for ubiquitylation, and the R I N G finger protein stabilizes the E2-cullin interaction. Two C D L s crucial for the cell cycle progression are the Skplp-cullin-F-box protein (SCF) complex or the anaphase promoting-complex/cyclosome ( A P C / C ) . Covalent attachment of a polyubiquitin chain on lysine residues o f the substrate mediates its recognition and subsequent degradation by the 26S proteasome. A t various points o f the cell cycle (e.g. S phase, metaphase), checkpoints exist and serve as surveillance mechanisms to ensure sequential execution o f events within the cell cycle, such that the execution o f a later event is dependent upon the completion o f a prior event (Hartwell and Weinert, 1989). In the event o f a spontaneous error or a failure to complete a step, activation o f a checkpoint causes transient arrest o f cell cycle  3  progression until the earlier event has been successfully completed, giving the cell more time to correct the error. Similar to the mitotic cell cycle, the fidelity o f D N A transfer in the germ-line during meiosis has to be precise for the maintenance o f a species. Meiosis I involves recombination (exchange o f D N A ) between homologous chromosomes and their segregation, whereas meiosis II, like mitosis, involves segregation o f sister chromatids. Defects in meiosis have devastating effects like miscarriage or birth defects, but a detailed discussion o f this topic is beyond the scope o f this thesis and w i l l not be elaborated upon further.  1.1.2 Budding yeast as a model organism to study the cell and chromosome cycle Since the cell cycle and chromosome cycle are basic and fundamental cellular processes, the mechanisms and genes involved are highly conserved among eukaryotes (reviewed i n (Chan et al., 2005; Kitagawa and Hieter, 2001)). Therefore, studies i n model organisms greatly facilitate the understanding o f normal human biology and mechanisms of human diseases. For instance, the baker's yeast Saccharomyces  cerevisiae has multiple  experimental advantages, including its short life cycle and ease o f genetic manipulation as either haploids or diploids, and the availability o f a battery o f powerful molecular and biochemical techniques. In addition, the cell cycle o f S. cerevisiae can be followed by cellular morphology because it divides by budding. Therefore, the size o f the daughter bud and the location o f nuclear D N A allow assessment o f the cell cycle stage within a population o f cells. For example, cells i n G l are unbudded; cells i n S phase are small budded; and those in G 2 / M are large budded (Figure 1.1). Indeed, Leland Hartwell, the 2001 Nobel prize laureate in Physiology and Medicine, identified key regulators o f the cell cycle i n the cell division cycle (cdc) mutant collection by isolating mutants that arrest at particular stages o f the cell cycle (Hartwell et al., 1974; Hartwell et al., 1970). H i s studies laid the foundation for our understanding o f the eukaryotic cell cycle. However, due to the small size o f budding yeast chromosomes, microscopic examination o f chromosome behaviors has traditionally been hindered by poor resolution. Cytological  4  studies i n larger eukaryotic cells have provided descriptions o f spindle dynamics and chromosome movements (Rieder and Salmon, 1994). More recently, elegant molecular genetic methods such as tagging chromosomes with green fluorescent protein (GFP) fused to a repressor, which binds to operator arrays integrated at a specific location in the genome, have allowed direct observation o f chromosome dynamics i n wild-type and mutant yeast strains (Straight et al., 1996). Studies i n different organisms thus complement each other and often reveal common, conserved cellular mechanisms. Phenotype screening based on marker stability i n budding yeast has provided a powerful approach for detecting and analyzing mutants i n genes that act to preserve genome structure. Several collections o f yeast mutants were isolated i n the last 2 decades by forward genetics (proceedings from phenotype to genotype) with the primary criterion of chromosome or plasmid loss, including the smc, mem, chl, cin and ctf collections (Hegemann et al., 1999; Hoyt et al., 1990; Kouprina et al., 1988; Larionov et a l , 1985; Larionov et al., 1987; Maine et al., 1984; Spencer et a l , 1990). MIF and CST genes are wild-type loci that induce chromosome instability when overexpressed (Meeks-Wagner et al., 1986; Ouspenski et al., 1999; Sarafan-Vasseur et al., 2002). Assays for gross chromosomal rearrangements (GCRs) have also been developed (Huang et al., 2003; M y u n g et al., 2001a; M y u n g et al., 2001b). M a n y o f the ede mutants also exhibit increased chromosome loss and/or mitotic recombination (Hartwell and Smith, 1985). Not surprisingly, different genetic screens have led to the identification o f different yet overlapping gene sets important for various steps i n the chromosome cycle, including proteins that function at the kinetochores, telomeres, origins o f replication, and i n microtubule dynamics, sister chromatid cohesion, D N A replication, D N A repair, D N A condensation and cell cycle checkpoints. Genes identified by this strategy have often supported successful identification o f functional homologues i n other eukaryotes. In 1996, S. cerevisiae became the first eukaryote to have its genome completely sequenced (Bassett et al., 1996; Goffeau et al., 1996), and has subsequently served as a test-bed for the development o f genomic, proteomic, bioinformatic and systems biology tools. These advances have greatly facilitated and accelerated the identification and  5  characterization o f genes important for chromosome maintenance. In the following sections, key cellular components and mechanisms pertinent to chromosome segregation in the budding yeast w i l l be discussed, and major differences with other eukaryotes w i l l be highlighted.  1.1.3 Biological processes that affect chromosome stability 1.1.3.1 Kinetochores mediate the attachment with mitotic spindles Centromere is the region o f D N A on a chromosome where the multiprotein kinetochore complex binds, and mediates chromosome-microtubule attachment. Interestingly, the C E N D N A size and composition vary greatly i n eukaryotes. The budding yeast C E N D N A consists o f only 125bp, with 3 conserved elements - the 8bp non-essential C D E I , the 78-86bp A T - r i c h C D E I I and the 25bp essential C D E I I I (Fitzgerald-Hayes et al., 1982) (Figure 1.2B). The C D E elements are flanked by highly phased nucleosome arrays for >2 kb (Bloom and Carbon, 1982). In contrast, the fission yeast S. pombe C E N D N A is more similar to higher eukaryotes C E N D N A in terms o f size and organization. Fission yeast C E N D N A is 35-100kb, consisting o f a 4-7kb central core o f non-repetitive sequence (cni) flanked by innermost repeats (imr) and outer repeats (ptr) (Figure 1.2B). Interestingly, some plants, insects and the nematode Caenorhabditis  elegans contain  holocentric chromosomes, where the kinetochores assemble all along the entire length o f the chromosome. Mammalian C E N D N A spans 2-4Mb, and is composed o f highly repeated a-satellite (171bp) D N A arrays (reviewed i n (Chan et a l , 2005; Kitagawa and Hieter, 2001; Pidoux and Allshire, 2005; Sharp and Kaufman, 2003; Yanagida, 2005)) (Figure 1.2B). The difference in C E N D N A sizes m a y b e related to the difference in chromosome size: budding yeast chromosomes are - 1 M b , whereas human chromosomes are ~150Mb. The increase in chromosome size i n mammals may require larger forces for chromosome movements. Indeed, one kinetochore of budding yeast binds only one microtubule, whereas one kinetochore o f fission yeast binds 2-4 microtubules, and one kinetochore o f higher eukaryotes binds 10-45 microtubules. Despite the differences i n C E N D N A size and the number o f microtubules binding to a kinetochore i n eukaryotes,  6  many kinetochore proteins and spindle checkpoint components are conserved (see below). This raises the possibility that metazoan kinetochores are assembled from repeated subunits, where each repeat might resemble the unit module o f the yeast kinetochore. W i t h advances i n experimental techniques, the list o f kinetochore-associated proteins in model organisms and human exploded i n recent years (reviewed i n (Chan et a l , 2005; Fukagawa, 2004; Houben and Schubert, 2003; M c A i n s h et al., 2003; Pidoux and Allshire, 2004; Yanagida, 2005)). To date, over 65 S. cerevisiae kinetochore proteins have been identified ( M c A i n s h et al., 2003), while the number o f the mammalian kinetochore proteins is predicted to be over 100 (Fukagawa, 2004). Kinetochore proteins are classified as structural or regulatory. Structural components physically bridge C E N D N A to spindle M T s ( M c A i n s h et al., 2003). Structural kinetochore components are further classified as inner, central, and outer kinetochore proteins based on their proximity to the C E N D N A (reviewed i n (Cheeseman et al., 2002b; M c A i n s h et al., 2003)). Inner kinetochore proteins interact with centromeric chromatin (e.g. Cse4p (yeast)/CENP-A (human) (see below)), while outer kinetochore mediates interaction with microtubules (e.g. the D a m l p / D A S H complex in yeast). Central kinetochore complexes (including the conserved, essential Ndc80p complex i n yeast and mammalian cells (Janke et al., 2001; Wigge and Kilmartin, 2001)) link the inner and outer layers. Regulatory proteins, including motor proteins, MT-associated proteins, regulatory proteins such as I p l l (yeast)/Aurora B (human) kinase, and spindle checkpoint components, function to regulate kinetochore-MT attachment and to co-ordinate events within the cell cycle (Biggins and Walczak, 2003; M c A i n s h et al., 2003). Centromeres o f higher eukaryotes are visualized as primary constrictions i n metaphase (Figure 1.2 A ) . O f particular note is the continued discovery o f the conservation o f individual kinetochore proteins and the overall organization o f protein complexes between higher eukaryotes and yeast. These findings support the concept that the basic building blocks o f kinetochores in these organisms may not be as different as first suspected based on the differences in underlying D N A sequence and size.  7  Despite that C E N D N A sequences vary among eukaryotes, C E N chromatin organization is conserved. D N A o f eukaryotic chromosomes is packaged into chromatin. The most basic level o f packaging involves 146bp o f D N A wrapping i n 1.75 turns around a nucleosome, which is composed o f an octamer o f core histones (2 o f each o f H 2 A , H 2 B , H3 and H4). A l l eukaryotes contain a centromere-specific nucleosomal structure, and the inner kinetochores contain a specialized histone H 3 , Cse4p (yeast)/CENP-A (mammals) (Stoler et al., 1995). Centromeres o f fission yeast and higher eukaryotes contain transcriptionally inactive heterochromatin and involve epigenetic control. Centromeric silencing in fission yeast and higher eukaryotes depends on the R N A interference machinery (Hall et al., 2002; Volpe et al., 2002), requires the histone methyltransferase C L R 4 / S U ( V A R ) 3 - 9 , which methlyates lysine 9 o f H 3 (Lehnertz et al., 2003), and the heterochromatin-binding protein S W I 6 / H P 1 , w h i c h binds to the trimethylated lysine 9 o f H 3 (Bannister et al., 2001; Lachner et al., 2001). Mutation o f either gene leads to chromosome instability (CIN) (Wang et al., 2000a). Mutation o f histone deacetylase (David et al., 2003) or o f its target H 3 also results i n improper establishment o f pericentric heterochromatin and leads to aneuploidy (Wei, 1999). Outer kinetochore proteins include microtubule-associated proteins ( M A P ) (e.g. D a m l p (yeast), B i k l p (yeast)/CLIP-170 (human), B i m l p (yeast)/EBl (human)) and motor proteins (e.g. C E N P - E (human), K i p 3 (yeast)ZMCAK (human), Cin8p (yeast)/BIMC (human), dynein), all o f which interact with microtubules (Heald, 2000; Hoyt and Geiser, 1996). Microtubules are hollow cylindrical tubes consisting o f a heterodimer o f a and P tubulins. Microtubules are polar molecules, with a dynamic 'plus' end and a 'minus' end at the microtubule organizing centre ( M T O C ) . The plus end, which w i l l capture kinetochores, undergoes rapid growth and shrinkage by polymerization and depolymerization, respectively, switching between these two states in events called "catastrophes" and "rescues" (Maddox et al., 2000). The minus side o f microtubules nucleates at the M T O C , which is called spindle pole body (SPB) i n yeast and centrosome i n higher eukaryotes. The centrosome is made up o f 2 barrel-shaped centrioles surrounded by a matrix o f pericentriolar material. The S P B is a disk-shaped  8  structure made up o f three plaques. Besides kinetochore microtubules, there are 2 other types o f microtubules: (1) interpolar microtubules which project towards the spindle midzone and interact with chromosome arms or overlap with microtubules emanating from the other pole, through the interaction o f micro tubule-associated factors and motors; and (2) astral (cytoplasmic) microtubules which project towards the cortex and are instrumental i n spindle orientation and positioning (Wittmann et al., 2001). Spindle disassembly is necessary for cytokinesis, and it is thought to occur b y depolymerization of the interpolar microtubules from their plus ends. Unlike other eukaryotes, the yeast nuclear membrane does not break down during mitosis, so the SPBs remain embedded within the nuclear membrane (Hoyt and Geiser, 1996). The nuclear and cytoplasmic faces o f the S P B are linked by a central plaque embedded in the nuclear envelope. During S phase, the S P B duplicates, while in prometaphase and metaphase, the SPBs separate (Winey and O'Toole, 2001). Initially, yeast sister kinetochores are both attached to one S P B , the " o l d " S P B , and this type o f kinetochore-microtubule attachment is called 'syntelic attachment' (Tanaka, 2002). The yeast kinetochores are positioned near the SPBs throughout the cell cycle (Jin et al., 2000). O n the other hand, kinetochore-microtubule interactions i n mammalian cells only take place during mitosis after the nuclear envelope breaks down. A t the onset o f mitosis, rapidly growing and shrinking microtubules probe the cytoplasm for kinetochores, i n a 'search and capture' mechanism that is stochastic and error-prone i n nature (Figure 1.3). During the early stage o f chromosome orientation, usually only one sister is attached to a pole, and this kind o f attachment is called 'monotelic attachment' (Figure 1.4C). Recently, mono-oriented chromosomes i n mammalian cells were shown to laterally interact with kinetochore microtubules o f bi-oriented chromosomes, which serve as tracks to help the mono-oriented chromosomes to 'hitch a hike' to the spindle equator (Kapoor et al., 2006). This interaction and chromosome movement is dependent on the mammalian kinesin-7 family member C E N P - E . This cooperative process increases the likelihood that mono-oriented chromosomes w i l l achieve bi-orientation because the middle o f the spindle is rich i n microtubules extending from the opposite spindle poles. If  9  both sister kinetochores attach to the same spindle pole (syntelic attachment, Figure 1.4B), the Ipllp/Aurora B kinase facilitates re-orientation by phosphorylating kinetochore targets (see below). The spindle checkpoint signal is maintained until sister kinetochores of all chromosomes bi-orient to opposite spindle poles, and this manner o f attachment is called 'amphitelic attachment' (FigureT.4A). When bipolar attachment is achieved, tension generated at the kinetochore by forces from opposite spindle poles and cohesin (see below) has a stabilizing effect on kinetochore microtubules (Ault and Nicklas, 1989; K i n g and Nicklas, 2000). Another type o f attachment error occurs when a single kinetochore becomes attached to microtubules from both spindle poles, which is called 'merotelic attachment' (Figure 1.4D). However, this defect is not detected by the spindle checkpoint (Cimini et al., 2001). Nevertheless, merotelic attachment rarely cause chromosome missegregation in mammals because the kinetochores usually make enough bipolar attachments to pull the sister chromatids to opposite poles. Interestingly, budding yeast chromosomes do not undergo congression to the metaphase plate (O'Toole et a l , 1999), but form two lobes that lie on either side o f the spindle midzone (Goshima and Yanagida, 2000; He et al., 2000). Both yeast and mammalian centromeric chromatin undergo transient separation before cohesion degradation at anaphase (Goshima and Yanagida, 2000; He et a l , 2000; Shelby et al., 1996; Tanaka et al., 2000).  1.1.3.2 M i t o t i c s p i n d l e c h e c k p o i n t  Spindle checkpoint components present at the kinetochore i n turn monitor M T attachment and/or tension and sense the completion o f metaphase, when bi-polar attachment o f all chromosomes has been achieved (Lew and Burke, 2003; Tanaka, 2002). BUB1 and 3 (budding uninhibited by benzimidazole) and MAD1, 2, and 3 (mitotic arrest deficient) are checkpoint genes first identified in yeast i n genetic screens that looked for mutants that fail to detect kinetochore-microtubule attachment errors caused by microtubule-depolymerizing drugs. A s a.result, these mutants do not arrest before anaphase despite the presence o f chromosomes not properly attached to the spindle, which leads to increased chromosome missegregation and increased sensitivity to  10  microtubule-depolymerizing drugs (Hoyt et al., 1991; L i and Murray, 1991; Weiss and Winey, 1996). These findings lead to the description o f a spindle checkpoint pathway that detects kinetochores that are not attached to microtubules or are not under tension ( Y u , 2002). Even a single unattached kinetochore can delay segregation o f already aligned chromosomes (Rieder et a l , 1995). Mammalian and yeast checkpoint proteins were shown to localize to kinetochores that have not yet attached to the mitotic spindle (reviewed in (Cleveland et al., 2003)). The exact sequence o f spindle checkpoint sensing and signaling is not completely understood, but probably involves amplification o f diffusible signals. BTJB1 is a kinase that is known to phosphorylate M A D 1 and B U B 3 , and B U B 3 i n turn binds to and activates B U B 1 . B U B R 1 is the mammalian homolog o f yeast Mad3p, but it has evolved to contain a kinase domain that is not present i n Mad3p. Localization o f B U B R 1 to the kinetochore is dependent on its interaction with B U B 3 , and B U B R 1 is postulated to act as a mechanosensor. Interaction o f B U B R 1 with the kinesin-like protein C E N P - E stimulates B U B R 1 kinase activity is stimulated (Chan et al., 1999; M a o et a l , 2003). C E N P - E is thought to act as a tension sensor and increases the efficiency o f microtubule capture; it is able to activate the spindle checkpoint i n the presence o f mono-oriented chromosomes (reviewed i n (Compton, 2006)). Yeast M p s l p (monopolar spindle) was originally identified to be involved i n S P B duplication, but was later found to have a role in the spindle checkpoint by phosphorylating M a d l p and recruiting other checkpoint components to unattached kinetochores (Weiss and Winey, 1996 (Winey and Huneycutt, 2002). M A D 1 binds to and recruits M A D 2 to the kinetochore (Chen et a l , 1999). M A D 2 binds to C D C 2 0 / S L P 1 / F I Z Z Y / P 5 5 ,  the substrate specificity factor o f the A P C / C ,  inhibiting its ubiquitin ligase activity ( Y u , 2002). Interestingly, M A D 1 and C D C 2 0 contain a similar domain to interact with M A D 2 , so their interaction with M A D 2 is mutually exclusive (Luo et al., 2002). M A D 1 - M A D 2 binding may catalyze a conformational change i n M A D 2 so that it is compatible for C D C 2 0 binding. M A D 1 hyperphosphorylation may be required to dissociate M A D 2 from M A D 1 for C D C 2 0 binding. B U B R 1 also directly binds C D C 2 0 and A P C / C components (Chan et al., 1999).  11  In addition, B U B R 1 forms a stoichiometric mitotic checkpoint complex ( M C C ) with B U B 3 , M A D 2 and C D C 2 0 (Sudakin et al., 2001). Vertebrate M A D 1 and M A D 2 are displaced from kinetochores with proper M T attachments. M A D 2 phosphorylation m a y b e involved i n silencing o f the checkpoint (Wassmann et al., 2003). The microtubule motor dynein has been implicated i n the highly dynamic turnover o f checkpoint components and i n checkpoint silencing. Checkpoint proteins like M A D 2 and B U B R l a r e thought to be released from the kinetochore through dynein-dependent transport v i a spindle M T s , and also v i a direct release o f proteins ((Howell et al., 2001); reviewed i n (Chan et al., 2005)). Z w l O , Z w i l c h and R o d which were first identified in Drosophila are also found i n higher eukaryotes but not in yeast. They form the R Z Z complex that is required for dynein localization ((Wojcik et a l , 2001); reviewed i n (Karess, 2005)). Even when kinetochore-microtubule connections are intact, a lack o f tension at the kinetochore can activate the checkpoint (Stern and Murray, 2001). The Aurora B/IPL1 kinase works with I N C E N P / S L I 1 5 as a tension sensor to promote turnover o f syntelic attachments; it works by destabilizing kinetochore-microtubule attachments through phosphorylation o f the microtubule-destabilizing mitotic centromere-associated kinesin ( M C A K ) i n vertebrate, analogous to D a m l p in yeast (Andrews et al., 2004; Cheeseman et al., 2002a; H e et al., 2001; K a n g et al., 2001; L a n et al., 2004; Stern and Murray, 2001; Tanaka, 2002). Aurora B may also phosphorylate B U B R 1 and M P S 1 for checkpoint signaling (Biggins and Murray, 2001). Aurora B / I P L 1 , I N C E N P / S L I 1 5 and SURVrvrN/BIRl are chromosomal passenger proteins that dynamically appear first in the inner centromere region between sister kinetochores, then move onto the elongating spindle in anaphase, and finally concentrate at the spindle midzone. Some structural kinetochore proteins (e.g. the N D C 8 0 complex) are also required for a functional checkpoint, which may first require the assembly o f a functional kinetochore (Gardner et a l , 2001; H e et al., 2001; Janke et al., 2001).  12  1.1.3.3 S i s t e r c h r o m a t i d c o h e s i o n  A t the end o f M phase in fission yeast and metazoan cells, or during late G l i n budding yeast, the cohesin complex, the "molecular glue" that holds sister chromatids together is loaded onto unreplicated D N A by the loading complex ( S C C 2 , S C C 4 ) (Ciosk et al., 2000). Cohesin is composed o f 4 subunits: S C C l ' / M C D 1 / R A D 2 1 , S C C 3 / I R R 1 ( S A I and S A 2 variants i n human), S M C 1 and S M C 3 (structural maintenance o f chromosomes). S M C 1 and S M C 3 contain globular ends with a hinge dimerization domain and a head A B C - t y p e ATPase domain, and a coiled-coil domain (Losada et al., 1998; Michaelis et a l , 1997) (Figure 1.5A). They form intra-molecular coiled coils by folding back on themselves, forming rod shaped proteins with the globular ATPase head at one end and the heterodimerization domain at the other (Haering et al., 2002). S M C 1 and S M C 3 dimerize through the hinge domain. The C-terminal and N-terminal ends o f S C C 1 bind to the head region o f S M C 1 and S M C 3 , respectively, and S C C 3 binds to the complex through S C C 1 (Haering et al., 2002; Haering et al., 2004). A T P hydrolysis is needed for cohesin loading onto D N A . , Cohesion is first established while sister chromatids are replicated i n S phase and is maintained until anaphase. A working model for cohesin is that it forms a ring structure that wraps around the sister chromatids in a topological association (Figure 1.5B). Cohesion is established along the whole length o f chromosomes, but is concentrated at the pericentromeric regions, spanning 50-60 kb, and at convergent transcription sites (intergenic A T - r i c h region) (Glynn et al., 2004). Kinetochores stimulate the recruitment o f cohesin, but this ability is not necessarily dependent on the centromere sequence per se (Megee et al., 1999; Weber et al., 2004). Cohesin recruited by the kinetochore may move to flanking regions, or kinetochores may influence surrounding chromatin to recruit cohesin. In S. pombe, the enrichment o f cohesin at peri-centromeric regions depends on the binding o f the H P 1-like protein SWI6 to histone H3 that is trimethylated on lysine 9 by C L R 4 (Bernard et al., 2001; Nonaka et al., 2002). In contrast, the nucleosome-remodeling complex R S C has been implicated i n the establishment o f chromatid arm cohesion only (Huang et a l , 2004; Huang and Laurent, 2004). Cohesin may also be redistributed to different places during transcription.  13  The establishment o f cohesion is not completely understood, but is thought to require the acetyltransferase E S C 0 1 / C T F 7 (Skibbens et al., 1999; Toth et a l , 1999), a variant replication factor C ( R F C - C T F 1 8 , C T F 8 , D C C 1 ) (Mayer et al., 2001; Mayer et al., 2004), a polymerase a-interacting protein C T F 4 (Hanna et al., 2001; Petronczki et a l , 2004), M R E 1 1 (Warren et al., 2004a), and the helicase C H L 1 (S, 2000; Skibbens, 2004). Additionally, P D S 5 is required to maintain cohesion at centromere proximal and distal sequences (Hartman et al., 2000). Cohesion sterically forces a back-to-back orientation to sister centromeres and promotes bi-orientation (Tanaka et al., 2000). Cohesion resists the force exerted by spindle microtubules emanating from opposite spindle poles on sister kinetochores, thereby generating tension (He et al., 2000). Cohesin is also recruited to double strand break ( D S B ) sites i n S / G 2 / M , and this recruitment requires S C C 2 (Strom et al., 2004; U n a l et al., 2004). Damage-induced cohesion may be important for D S B repair by holding broken ends close to homologous sequences, thereby facilitating homologous recombination. In budding yeast, cohesin remains associated with whole chromosomes until anaphase, whereas in mammalian cells, cohesins dissociate from chromosome arms in prophase in a Polo-like kinase 1 (PLKl)-dependent manner (Hauf et al., 2005). P L K 1 is activated as C D K levels rise at the onset o f M phase, and P L K 1 promotes arm cohesin dissociation through phosphorylation o f the S C C 3 - l i k e subunits, S A 1 and S A 2 . However, cohesin at centromeres persists until anaphase. This retention is dependent on shugosin ( S G 0 1 / M E I - S 3 3 2 in Drosophila)  (Hauf et al., 2005). Protein phosphatase 2 A (PP2A)  associates with S G O l and is required for protection o f centromeric cohesion by dephosphorylation o f cohesin (Kitajima et al., 2006; Riedel et al., 2006). Before the onset of anaphase, an inhibitory chaperone, securin/PDSl, binds to the separase/ESPl, thereby inhibiting it but also priming its activity, possibly by promoting its nuclear localization or protecting it from degradation (Ciosk et al., 1998). When all chromosomes align at the metaphase plate, A P C / C  C D C 2  ° targets securin for degradation (Cohen-Fix et al., 1996).  Separase is then released, and it cleaves the cohesin subunit S C C 1 , leading to the  14  breakdown o f cohesion and the beginning o f anaphase where sister chromatids move to opposite spindle poles (Michaelis et al., 1997; Uhlmann et al., 1999; Uhlmann et al., 2000) (Figure 1.6). P L K 1 phosphorylation o f S C C 1 enhances its cleavage by separase. A P C / C also degrades cyclins, lowering C D K activity and promoting exit from M phase. In meiosis, sister chromatids segregate to the same pole during meiosis I and b i orientation o f sister chromatids is suppressed by monopolin ( M A M 1 , C S M 1 , and L R S 4 ) (Toth et al., 2000). The SCC1 subunit o f the meiotic cohesin complex is replaced by R E C 8 . The paired homologous chromosomes are held together at chiasmata that are formed during recombination. In meiosis I, R E C 8 present on chromosomal arms is cleaved, thereby resolving chiasmata, while centromeric R E C 8 is protected during meiosis I by S G O l . In anaphase I, homologous chromosomes segregate to opposite poles (Buonomo et al., 2000; K l e i n et al., 1999). Meiosis II resembles mitosis with sister chromatids segregating to opposite poles.  1.2 Chromosome instability (CIN) and cancer 1.2.1 Aneuploidy is a hallmark of cancer Two types o f genetic instability are observed i n cancers: (1) instability at the nucleotide level, especially at microsatellite repeats ( M I N , microsatellite instability) and (2) instability involving whole chromosomes or large portions o f chromosomes ( C I N , chromosomal instability). The majority o f solid tumors exhibit genomic instability at the chromosomal level (Rajagopalan et al., 2003) (e.g. 85% colon cancers exhibit C L N and 15% exhibit M I N ) , and the occurrence o f M I N and C L N usually does not overlap. M I N tumors exhibit a 1000-fold increase i n point mutation rate, i n particular accumulation o f length alterations i n simple repeated sequences (units o f l-3bp), whereas C I N tumors exhibit increased rates o f chromosome missegregation, leading to the generation o f aneuploid cells. Changes in whole chromosome number or structural rearrangement o f chromosomes are commonly observed i n tumors (Cahill et al., 1998; Rajagopalan et al., 2003). Large-scale chromosomal gains or losses can be detected by flow cytometry in a  15  cell population, and chromosomal rearrangements (>1 M b ) i n an individual cell can be revealed by comparative genomic hybridization ( C G H ) , multiplex fluorescence in situ hybridization ( M - F I S H ) or spectral karyotyping ( S K Y ) . Aneuploidy and chromosomal rearrangements may play a role i n tumor progression by causing an imbalance i n the dosage o f many genes at once. For instance, chromosome loss or partial chromosomal deletion results i n loss o f heterozygosity ( L O H ) , which can lead to reduced expression o f tumor suppressor genes located in the region, or uncover recessive mutations in the remaining allele. Chromosome gain or partial amplification can amplify oncogenes within the region. Overexpression o f oncogenes and/or reduced expression o f tumor suppressor genes would create a growth advantage through increased proliferation or reduced cell death, and result in clonal expansion. This scenario would repeat for each new growth-promoting mutation, and constitutes the basis o f the theory o f multi-step carcinogenesis (Boland and Ricciardiello, 1999). Identifying recurrent chromosomal aberrations at specific loci i n cancer cells may provide clues for the identification o f oncogenes and tumor suppressor genes. A n average cancer o f the colon, breast, pancreas or prostate loses 25% o f its alleles on a chromosome (Lengauer et al., 1998). Some primary breast cancers exhibit >20 regions with L O H when analyzed with microsatellite markers (reviewed in (Loeb, 2001)). Interestingly, analysis o f polymorphic markers in 5 chromosomes in colorectal cancer cell lines indicated that mechanisms underlying L O H were chromosome-specific. Partial losses were predominant for some chromosomes, while whole chromosome losses were responsible for others. For partial loss, gross chromosomal rearrangement ( G C R ) , not mitotic recombination, was the predominant mechanism. For whole chromosome loss, mitotic nondisjunction was responsible, and reduplication o f the remaining chromosome was followed i n some cases. L O H occurs at different frequency at different regions o f each chromosome, implying that L O H is coupled with clonal selection for loss o f tumor suppressor genes (Thiagalingam et al., 2001).  16  1.2.2 Relationship between a state of aneuploidy and an increased rate of chromosome instability (CIN) Observation o f a state o f aneuploidy in cancer cells does not directly imply an increased rate o f C I N , because aneuploidy may be caused b y factors other than C I N . For instance, aneuploidy can be caused b y chromosome missegregation i n a single cell division (at a normal rate), followed b y clonal expansion o f the aneuploid cell due to some selective advantage; or, the survival o f an aneuploid cell can result from a defect i n the apoptotic pathway. However, an analysis o f 98 aneuploid gastric tumors b y F I S H and flow cytometry showed intratumoral variations i n chromosome copy number; this population heterogeneity suggests that aneuploidy is associated with C I N (Furuya et al., 2000). In another study, 16 out o f 25 pancreatic carcinomas showed karyotypically related clones, signifying monoclonal origin and evolutionary variation (Gorunova et al., 1998). Similarly, F I S H analysis o f aneuploid colorectal cancer cell lines for 6-7 generations showed that losses or gains o f chromosomes occurred at >10" per 2  chromosome per generation, which is 10-100 times more often than i n diploid cancers o f the same histological subtype (Lengauer et al., 1997). These observations are consistent with the hypothesis that aneuploidy i n cancers is caused b y C I N . T o further delineate the relationship between C L N and aneuploidy, Lengauer et al. introduced an extra chromosome into a diploid cell line and fused two diploid lines to artificially create aneuploid cell lines. These lines, unlike natural C L N tumor lines, did not display C I N , suggesting aneuploidy per se does not cause C I N (Lengauer et al., 1997).  1.2.3 CIN occurs at early stage of cancer, and can be a driving force in tumorigenesis The timing o f C L N occurrence during tumorigenesis, and the role o f C L N i n tumorigenesis have been highly debated. One hypothesis postulates that for a cancer cell to accumulate the 6-10 genetic alterations required for its proliferation and survival, it must be genetically unstable, thereby suggesting that genetic instability occurs at the early stage o f cancer, and represents an important step i n the initiation and/or progression  17  of tumorigenesis (Davies et al., 2002; Hartwell et al., 1997; Parsons et al., 2005). In support o f this hypothesis, aneuploidy has been observed i n small benign colorectal tumors and uterine leiomyomas (El-Rifai et al., 1998), and >90% o f early colorectal adenomas studied (1-3 m m in size) have allelic imbalance (Bardi et al., 1997; Bomme et al., 1998; Lengauer et al., 1998; Shih et al., 2001). The prevalence o f aneuploidy i n benign colorectal tumors is less than that i n cancers, but the deviations from a normal karyotype increase as the tumors enlarge in size (Bardi et al., 1997; Shih et al., 2001). Aneuploidy is associated with poor prognosis and correlates with the severity o f the disease (Rajagopalan and Lengauer, 2004a). C I N may serve as an engine o f both tumor progression and heterogeneity (Jallepalli and Lengauer, 2001; Vogelstein and Kinzler, 2004).  1.2.4 G e n e t i c b a s i s o f C I N i n c a n c e r 1.2.4.1 C a n c e r - p r o n e s y n d r o m e s  Germline mutations causing genomic instability, particularly i n genes involved in D N A damage recognition and repair, are now recognized as being important predisposing conditions for cancer (reviewed i n (Hoeijmakers, 2001; Levitt and Hickson, 2002; Vogelstein and Kinzler, 2004)). For instance, the less common M I N phenotype i n colorectal cancer was first described i n 1992. The similarity o f phenotype i n MUST tumor cells and D N A mismatch repair ( M M R ) mutants i n yeast and E. coli rapidly led to the identification o f mutations in M M R genes (based on a candidate gene approach) in hereditary nonpolyposis colon cancer ( H N P C C ) , which account for 3% o f colon cancer (Fishel et al., 1993; Leach et a l , 1993; Papadopoulos et al., 1994; Strand et al., 1993). Another example is provided by xeroderma pigmentosum ( X P ) patients whose cells have defects i n the nucleotide excision repair ( N E R ) pathway and high mutation rates due to pyrimidine dimers; these patients develop skin cancers at high rates. Table 1.1 summarizes germline mutations in genes involved i n maintaining genomic integrity that are known to underlie cancer-prone syndromes, and lists the function/pathway o f the encoded protein, evolutionary conservation between yeast and human genes, and the  18  mode o f inheritance o f the diseases. These "caretaker" genes, unlike conventional oncogenes and tumor suppressor genes which directly control cell birth and death, affect the integrity o f the genome and control the mutation rate. Interestingly, despite the ubiquitous expression o f these genome maintenance proteins, mutations in these genes lead to tissue-specific tumor predispositions. In addition, somatic mutations i n these same genes may not occur i n sporadic tumors o f the same type (Sieber et a l , 2003). In fact, although tumor types from one specific organ have a tendency to share mutations in certain genes or i n different genes within a single pathway, they rarely have uniform genetic alterations, demonstrating the heterogeneous nature o f cancer (Boland and Ricciardiello, 1999). In contrast to M I N , the genetic basis o f the commonly observed C L N i n sporadic cancers is not well understood. Cytologically, many cancer cells exhibit aberrant cell architecture, including abnormal centrosomes, multipolar spindles, and breakage-fusionbridge cycles (Gisselsson, 2003; Saunders et al., 2000). Intuitively, C I N , and therefore aneuploidy, can be caused by errors i n chromosome segregation. M a n y cellular mechanisms are responsible for proper chromosome transmission, such as D N A replication, sister chromatid cohesion, centrosome duplication and segregation, kinetochore-microtubule attachment, mitotic spindle checkpoint, D N A condensation, D N A repair and cytokinesis (Figure 1.7). One approach to determine the genetic basis o f CLN in tumors is to identify mutations i n genes known to be important for chromosome segregation in human cells, or in human homologues o f C I N genes discovered in model organisms, which serve as cross-species candidate C L N genes.  1.2.4.2 M u t a t i o n s i n m i t o t i c s p i n d l e c h e c k p o i n t  M a n y C L N genes were originally identified and studied i n model organisms such as yeast, and later found to have conserved functions and cancer relevance, including the genes listed in Table 1.1 and mitotic spindle checkpoint components. One important class of cancer relevant genes first discovered i n yeast are the spindle checkpoint proteins, which monitor kinetochore-MT attachment and alert the cell to potential chromosome  19  segregation errors by specifically binding to kinetochores that have not attached to M T s . BUB1 and BUB IB (encoding BTJBR1) are mutated i n colorectal tumors and several other cancer types at a low frequency (Cahill et al., 1998; Gemma et al., 2000; Ohshima et al., 2000; R u et al., 2002; Shichiri et a l , 2002) (Table 1.2). Epigenetic silencing through promoter hypermethylation o f BUB1 and BUB IB has also been found in aneuploid colon carcinoma (Shichiri et al., 2002). The recent report that germline biallelic mutations in the spindle checkpoint gene, BUB1B, is associated with mosaic variegated aneuploidy ( M V A ) and inherited cancer predispositions strongly supports a causal link between C L N and cancer development (Hanks et al., 2004). Human homologues o f other yeast mitotic checkpoint proteins (MAD1, 2, 3 and BUB1, 3) then became candidate C I N genes and were subsequently tested for mutations i n tumors. MAD2 is mutated in gastric cancers ( K i m et al., 2005), and downregulated i n cancer cell lines ( L i and Benezra, 1996; M i c h e l et al., 2001; Wang et al., 2002b). However, no mutation i n other spindle checkpoint genes was found (Cahill et al., 1999), suggesting they could be altered by misregulation or that other C L N genes could be affected. For example, MAD1 binds to the Tax oncoprotein from the human T-cell leukaemia virus type 1 ( H T L V - 1 ) and prevents MAD2 activation (Jin et al., 1998). L A T S 1 / W A R T S (large tumor suppressor homologue 1), a paralog o f B U B 1 , is a mitosis-specific serine/threonine kinase that interacts with M O B 1 ( M p s l - O n e binder) and may play a role i n the mitotic exit network, cytokinesis, and coordination between cell proliferation and apoptosis. Downregulation o f L A T S 1 has been found to contribute to tumor formation (Bothos et al., 2005; Hergovich et al., 2006; L a i et al., 2005; Y a n g et al., 2004). The recent survey o f C L N colorectal tumors for mutations in 100 human homologues o f C I N genes identified i n yeast and flies, including 6 kinetochore/spindle checkpoint proteins, represents a stunning proof o f principle: Wang et al. identified.5 new C L N cancer genes, including the kinetochore/spindle checkpoint genes Rod, ZwlO, and Zwilch (Table 1.2), which together account for - 2 % o f the mutational spectrum i n colorectal cancers (Wang et al., 2004b). These proteins function together as the R Z Z complex to recruit the dynein-dynactin complex and M A D 1 - M A D 2 to the kinetochore. The R Z Z complex is thought to have a role i n spindle checkpoint  20  activation and inactivation (reviewed in (Karess, 2005)). The infrequent mutation rate in spindle checkpoint genes raises the possibility that C L N i n cancer cells could be caused by mutation o f any one o f many genes involved i n chromosome segregation, including other kinetochore proteins. Because o f the large number o f candidate genes that could be mutated to give a C L N phenotype, the frequency o f a particular mutation may be low, as is observed for the spindle checkpoint genes. Interestingly, analysis o f the mitotic index o f cancer cell lines i n response to microtubule-disrupting reagents showed that the mitotic spindle checkpoint is often impaired, but not completely absent (Gascoyne et al., 2003; Saeki et al., 2002; Takahashi et al., 1999). Absence o f the checkpoint proteins M A D 2 , B U B 3 , or B U B R 1 i n mice and C. elegans yields early embryonic lethality (Babu et al., 2003; Baker et al., 2004; D a i et al., 2004; Kalitsis et al., 2000; Kitagawa and Rose, 1999; K o p s et al., 2004; M i c h e l et al., 2001). Mouse models o f defective checkpoints, where a checkpoint component is reduced in concentration, result i n a small increase i n cancer susceptibility. For example, mice heterozygous for BUB IB or BUB 3 are more prone to colorectal or lung tumors after challenge with carcinogen (Babu et al., 2003; D a i et al., 2004). R A E 1 , which has homology to B U B 3 , mediates nuclear export o f m R N A through nuclear pores during interphase and binds to B U B 1 at kinetochores during mitosis. Heterozygous RAE1 mice have increased aneuploidy and develop lung tumors at an increased rate (Babu et al., 2003). 28% o f heterozygous MAD2 mice develop lung tumors at high rates after long latencies (Babu et al., 2003; Baker et al., 2004; D a i et al., 2004; Kitagawa and Rose, 1999; Kops et al., 2004; M i c h e l et al., 2001). Additionally, some tumor suppressor genes affect the levels o f checkpoint components at the transcript level. For example, B R C A 1 regulates MAD2 transcript levels directly by binding to its promoter, and mouse cells that express mutant BRCA1 have decreased expression o f M A D 2 , B U B 1 , B U B R 1 and Z w l O (Wang et al., 2004a). A single nucleotide polymorphism i n MAD1 that affects M A D 2 binding and recruitment o f M A D 2 to kinetochores has recently been found i n a breast cancer cells (Iwanaga et al., 2002). These results suggest that biallelic expression o f  21  checkpoint components is important for their function, and a weakened checkpoint might facilitate tumorigenesis.  1.2.4.3 M i s r e g u l a t i o n o f k i n e t o c h o r e p r o t e i n s  Mutations i n genes encoding structural kinetochore proteins have not yet been identified i n cancer cells, possibly because most have not been examined. Since 5 out o f 8 (BUB1, BUBR1, Rod, ZwlO, Zwilch, CDC4, MRE11A,  and Ding) C I N genes known to  be mutated i n C L N colon cancers encode kinetochore or spindle checkpoint proteins (Cahill et al., 1998; Rajagopalan et al., 2004; Wang et al., 2004b), the kinetochore offers a logical choice for mutational testing. Furthermore, the - 1 0 0 predicted human genes that encode kinetochore components comprise a large mutational target that could be mutable to a C L N phenotype (Fukagawa, 2004). For example, kinetochore proteins constitute a significant portion o f the collection o f chromosome transmission fidelity (ctf) mutants identified i n a classical genetic screen i n yeast (9 out o f the 24 C T F genes cloned and characterized to date; see Table 2.1) (Spencer et al., 1990). Systematic mutational analysis o f kinetochore genes i n various cancers would shed light on the frequency o f specific mutations i n kinetochore genes and their potential role i n tumorigenesis. On the other hand, expression studies have suggested a correlation between overexpression o f several kinetochore proteins and cancer (Table 1.2). C E N P - A is overexpressed and mistargeted i n colorectal cancer tissues (Tomonaga et al., 2003). Overexpressed C E N P - A localizes to the entire chromosome and dissociates from native centromeres. This causes a subset o f kinetochore proteins to be recruited to noncentromeric chromatin, leading to ectopic formation o f pre-kinetochore complexes, potentially depleting some kinetochore components, thereby disrupting the native centromere-kinetochore complex and causing C I N (Van Hooser et al., 2001). Another inner kinetochore protein, C E N P - H , which is important for kinetochore organization, is also upregulated in colorectal cancer tissues (Tomonaga et al., 2005). Transfection o f a C E N P - H expression plasmid into diploid cell lines induces aneuploidy and increases the incidence o f aberrant micronuclei, suggesting that upregulation o f C E N P - H can lead to a  22  CLN phenotype. In addition, Aurora B (ALM-1) and I N C E N P , two chromosome passenger proteins that localize to the kinetochore from prophase to metaphase and to the mitotic spindle i n cytokinesis, are upregulated i n tumor cell lines (Adams et al., 2001; Sorrentino et a l , 2005; Tatsuka et al., 1998). Aurora B phosphorylation is required for chromosome condensation, controlling M T dynamics including destabilizing syntelic M T attachments to kinetochores, and regulation o f cytokinesis (reviewed i n (Giet et al., 2005)). Aurora B-overexpressing cells exhibit C L N and contain multinuclei, and injection o f these cells into nude mice induces tumor growth (Ota et al., 2002; Sorrentino et al., 2005). Conversely, a block o f Aurora B expression increases the latency period and reduces the growth o f thyroid anaplastic carcinoma cells (Sorrentino et al., 2005), supporting a causative link between Aurora B expression and cancer initiation or progression. Similarly, overexpression o f C E N P - F (mitosin) correlates with tumor proliferation and metastasis; hence, C E N P - F is suggested to be a potentially valuable proliferation marker for diagnosis and prognosis (Clark et al., 1997; de la Guardia et al., 2001; Erlanson et al., 1999; Esguerra et al., 2004; L i u et al., 1998; Shigeishi et al., 2005). C E N P - F is a cell cycle-regulated protein that associates with the outer kinetochore in M phase and is rapidly degraded upon completion o f mitosis. It associates preferentially with kinetochores o f unaligned chromosomes, and may play a role i n the spindle checkpoint (Chan et al., 1998; Yang et al., 2005; Y a n g et al., 2003). The evidence above suggests that overexpression o f kinetochore components may contribute to tumor progression by driving CLN. Stoichiometric expression o f kinetochore components may be important for functional kinetochore assembly and the dosage may be crucial for spindle checkpoint signalling. However, it is possible that overexpression is a consequence rather than a cause o f dysfunctional cell cycle regulation i n carcinogenesis. To delineate the causal relationship between kinetochore protein mutation/misregulation and cancer development, further functional studies must be performed in diploid cell lines or mouse models to investigate whether kinetochore mutation/misregulation leads to C L N or cellular transformation.  23  1.2.4.4 Additional examples of mutations in genes involved in chromosome segregation Systematic mutation testing o f candidate C I N genes i n colorectal cancer also identified somatic mutations in CDC4, MRE11A,  and Ding (Rajagopalan and Lengauer,  2004b; Wang et a l , 2004b). K n o w n mutations together account for only - 2 0 % o f the CLN mutational spectrum o f colon cancer. C D C 4 is a conserved F-box protein that functions in the S C F E3 ubiquitin ligase, involved in regulating the G l - S cell cycle checkpoint. C y c l i n E , an oncoprotein and a known target o f C D C 4 in mammalian cells, is overexpressed when C D C 4 is defective (Rajagopalan et al., 2004; Strohmaier et al., 2001). M R E 1 1 A is involved i n sister chromatid cohesion and D S B repair. Germline mutations i n M R E 1 1 A are responsible for ataxia telangiectasia-like syndrome (see Table 1.1). D i n g is uncharacterised, but its C-terminus is homologous with the yeast securin, PDS1 (Wang et al., 2004b). The human securin, also known as pituitary tumor transforming gene 1 (PTTG1), is overexpressed i n some cancers and its expression level is correlated with the invasiveness (Pei and Melmed, 1997; Zhang et al., 1999; Z o u et al., 1999). Aurora A kinase ( S T K 1 5 / B T A K ) at the centrosome is amplified and overexpressed in cancers (Zhou et al., 1998), and is associated with centrosome amplification, tetraploidization and aneuploidy. Indeed, approximately 80% o f invasive tumors show centrosome abnormalities i n size and number, and a significant proportion o f solid tumors are tetraploid, such as i n Barrett's oesophagus and ulcerative colitis (Rajagopalan and Lengauer, 2004a). Familial polyposis coli ( F A P ) patients and over 85% o f colorectal tumors have somatic mutations o f adenopolyposis coli (APC) (see Table 1.1), and this is the earliest event in sporadic colorectal tumor. Most APC mutations lead to loss o f the C-terminal domain that interacts with microtubules (and binds components o f checkpoint), failure to degrade beta-catenin, and have been postulated to contribute to C L N (Fodde et al., 2001a; Fodde et al., 2001b; Green and Kaplan, 2003; Kaplan et al., 2001). However, some cells with APC mutations undergo polyploidization i n whole-genome increments instead o f  24  aneuploidy, and some M I N cell lines with APC mutations remain diploid, so the exact significance of APC mutation i n C L N is still unclear (Fodde et al., 2001b). The genetic basis for C L N is just beginning to be understood (Figure 1.8). The daunting task o f screening the remaining hundreds o f candidate C I N genes lies ahead. Systematic mutation screening in candidate genes in signalling pathways have yielded success (Davies et al., 2002; Parsons et al., 2005). However, mutations i n C I N genes could be functional (leading to CLN) or merely "passenger" mutations that accompany tumorigenesis. The prevalence o f point mutations i n sporadic C I N colorectal cancers was determined to be approximately one nonsynonymous somatic change per M b o f tumor D N A , which is consistent with a rate o f mutation i n normal cells (Wang et al., 2002a). These data suggested that most sporadic C L N colorectal cancers do not display M I N or instability at the nucleotide level (Wang et al., 2002a). These results have significant implications for the interpretation o f somatic mutation observations i n candidate tumorsuppressor genes, suggesting these are likely to be o f functional relevance.  1.2.4.5 T h e r a p e u t i c i m p l i c a t i o n s  Designing effective therapeutics for cancer w i l l rely first on understanding the genetic basis o f cancer, including the cause o f C L N and its contribution to human cancers. This w i l l involve identifying the mutational spectrum and analyzing expression profiling of candidate C L N genes, and determining their functional consequence. Such knowledge could have several important practical applications. First, it would allow subclassification o f tumors based on the specific C L N gene mutation or misregulation, which could have implications for improved diagnostics, prognosis, or predictions o f response to therapy. For example, overexpression o f either Aurora A or B kinases causes C L N . Inhibition o f aurora kinases results i n a 98% reduction i n tumor volume i n nude mice injected with human leukemia cells (Harrington et al., 2004). One complication in studying cancer is that cancer is a heterogeneous disease, with many different genes mutated at low frequencies in different tumors and i n sub-population o f cells within individual tumors. Genetic instability is expected to contribute to heterogeneity.  25  However, i f a defined subset o f C I N genes represents the major C L N mutational targets in cancer, they may provide a rationale for therapeutic design to selectively k i l l tumor cells carrying C L N mutations (Hartwell et al., 1997; Hartwell and Weinert, 1989). While C I N may be important in the development o f a tumor, understanding the genetic and phenotypic differences between C I N tumor cells and normal cells may define an "Achilles heel" i n C L N tumors (relative to adjacent normal tissue), allowing selective killing o f tumor cells (Hartwell et al., 1997; Hartwell and Weinert, 1989). One approach is to identify drug targets that are specifically present and essential for the viability o f cancer cells, but are not present i n normal cells. For instance, fusion oncoproteins are generated by cancer-associated chromosomal translocations, such as the . fusion o f the breakpoint cluster region ( B C R ) with Ableson murine leukemia viral oncogene homologue ( A B L ) i n chronic myelogenous leukaemia ( C M L ) . However, it is difficult to identify drugs that can discriminate a protein between its normal and pathogenic state. Imatinib mesylate (produced by Glivec) inhibits both B C R - A B L and A B L , and several other kinases. O n the other hand, a drug screening strategy aimed at restoring the function o f tumor suppressor genes and defective apoptotic pathways, though genetically different i n tumor and cancer cells, might turn out to be a suboptimal approach, because it w i l l be unlikely to identify drugs that can reactivate genes/proteins to restore normal protein function (Sager and Lengauer, 2003). Context-driven therapeutics depend on the identification o f conditions i n which the requirement for a particular target is enhanced i n the context o f cancer cells compared with normal cells, which can be due to intrinsic (e.g. genetic or epigenetic) or extrinsic (microenvironmental) changes, or both. Most anticancer drugs i n use today affect targets present in both normal and cancer cells (Kaelin, 2005). One scenario o f differential requirements that can be exploited is the phenomenon o f synthetic lethality (SL). Synthetic lethality occurs when mutations i n two different genes, while non-lethal as mutations, become lethal when combined in a cell as a double mutant. B y targeting a specific gene in a cancer cell containing another known mutation could lead to synthetic lethality and selective killing. In this regard, an on-going effort i n model organisms such  26  as yeast has been to construct a comprehensive synthetic lethal genetic interaction map. B y definition, these second-site loss-of-function mutations (which are otherwise nonlethal in the CIN-gene wild-type cells) define proteins that, when reduced i n activity, cause lethality in the reference C I N mutant. I f the synthetic lethal interactions are conserved in humans, these second-site genes may suggest cross-species candidate proteins i n humans that when inhibited (e.g., by a drug) would specifically k i l l tumor cells relative to normal cells. Synthetic lethal interactors that are common to multiple CLN mutants may suggest candidate drug targets that are effective for selective killing C I N cancers with different C I N gene mutations. I f kinetochore proteins, for example, turn out to represent a significant fraction o f the C I N mutational spectrum in cancer, it is conceivable that second-site genes w i l l exist that are synthetically lethal i n combination with an entire set o f the kinetochore gene mutations, and therefore provide common drug targets for killing a broad spectrum o f C I N cancers. While the relevance o f kinetochore dysfunction to cancer still needs to be verified, defects i n human mismatch-repair genes, MLH1,  MSH2, and PMS2, are known to confer predisposition to colon cancer. Synthetic  lethality data i n yeast show that they are lethal i n combination with mutations i n D N A polymerase 5 and s that are otherwise viable. These latter enzymes catalyze D N A replication, and i n the process they proofread the growing strand o f D N A for errors. The results in yeast reveal the possibility o f selectively killing M I N cancer cells by interfering with D N A polymerases (reviewed i n (Friend and Oliff, 1998)). Recently, R N A interference screens have been applied i n mammalian cells to decipher synthetic lethality relationships and identify novel targets (Ngo et al., 2006; Willingham et al., 2004) (reviewed i n (Brummelkamp and Bernards, 2003)). The selective killing concept can be expanded to synthetic dosage lethality where one loss-of-function mutation causes lethality when combined with overexpression o f another protein. In therapeutics, loss o f function o f the second gene can be caused by drug inhibition. One example is that inactivation o f retinoblastoma protein (RB) in cancer leads to an increase in E 2 F activation, which in turn activates various genes involved in S-phase entry, including topoisomerase 2. Topoisomearse II inhibitors such as etoposide  27  bind to topoisomerase II, causing D N A strand breaks and apoptosis. Therefore, RBpathway mutations sensitize cells to topoisomerase II inhibitors. In a similar way, phosphatase and tensin homologue ( P T E N ) tumor-suppressor protein negatively regulates the phosphatidylinosital 3-kinase (PI3K) pathway, and a mammalian target o f the rapamycin (mTOR) pathway. PTEN~'~ cells are reported be more sensitive to the antiproliferative effects o f m T O R inhibitors than wild-type cells (Neshat et al., 2001). Defective mechanisms to maintain genomic stability render most cancers more vulnerable to genotoxic challenges. For example, XP mutations cause sensitivity to U V radiation, and mutations i n ATM and BRCA2 cause sensitivity to ionizing radiation (IR). In addition, caffeine inhibits A T R , and can induce S-phase cells to undergo premature chromosomal condensation. Cells lacking P53 i n G I control are found to be more susceptible to caffeine treatment. To identify the target pathway o f anticancer drugs, the differential sensitivity o f isogenic yeast mutants, each defective i n a particular D N A repair or cell cycle checkpoint function, to Food and Drug Administration ( F D A ) approved cytotoxic anticancer agents, such as cisplatin, camptothecin, and hydroxyurea, were tested (Dunstan et al,,2002; Hartwell et al., 1997; L u m et a l , 2004; Simon et al., 2000). Similar drug sensitivity assays can be set up in matched pairs o f cell lines (which differ in one genetic alteration) and in tumor cell lines (Dolma et al., 2003; Hartwell et al., 1997; Torrance et a l , 2001). Another avenue o f cancer therapeutics is to activate the cell cycle checkpoint, arrest cells and induce apoptosis. Taxanes (stabilizing microtubules) and vinca alkaloids (inhibiting microtubule assembly) are used to treat breast and ovarian cancer patients, and they reduce tension or produce unattached kinetochores i n mitosis by altering microtubule dynamics and cause long-term mitotic arrest. It is suggested that cells exit long-term mitotic arrest (through adaptation), but then apoptosis is induced i n ' G l (Tao, 2005). A n inhibitor o f K S P / E G 5 , a kinesin MT-dependent motor, is under phase I and II clinical trials and has the advantage that it only affects dividing cells because K S P only functions in mitosis. However, cells with a weakened checkpoint are less sensitive to microtubule-targeted drugs.  28  Paradoxically, complete inhibition o f the mitotic checkpoint can also be effective in cancer therapeutics. Reducing M A D 2 or B U B R 1 to <10% level i n various tumor cell lines causes complete inactivation o f the mitotic checkpoint and results in massive chromosome misdistributions, and lethality results i n 2-6 cell division (Kops et al., 2004; M i c h e l et al., 2004). This is probably because the rate o f chromosome missegregation is elevated to such high level that is incompatible with cell viability. A s mentioned above, MAD and BUB genes were indeed first identified as mutants defective i n triggering cell cycle arrest in response to microtubule inhibitors. If drugs could be used as tools to identify genes involved i n a related process, we should be able to use C I N genes to identify new anticancer drugs and better understand their modes o f action. Whether genomic instability reflects cause or effect o f altered cell physiology during tumorigenesis, a comprehensive identification o f genes whose mutation leads to chromosome instability is an important* but daunting, goal yet to be achieved. Understanding the etiology and tolerance o f genome instability i n viable cells is fundamental to understanding the development and survival o f cancers, and may be instrumental i n the design o f therapeutic approaches that take advantage o f specific vulnerabilities exhibited by cancer cells.  1.3 O v e r v i e w o f thesis  The goal o f m y thesis was to systemically identify functional determinants required for mitotic chromosome transmission in yeast, and extend the investigation to human cells based on cross-species protein sequence comparison. This analysis provided candidate C I N genes for cancer mutation testing i n cancer patients. The results w i l l be directly relevant to understanding o f cancer development, and may be useful i n developing strategies for cancer therapy and for sub-classification o f tumors based on their CLN mutational spectrum (Figure 1.9). Chapter 2 describes the systematic identification o f non-essential yeast genes important for the maintenance o f chromosome stability using multiple assays. The comprehensive C I N gene set includes expected and unexpected genes, providing not only  29  a rich resource for the study o f mechanisms required for accurate chromosome transmission, but also a list o f candidate human C I N genes based on protein sequence similarities. Examples o f known C L N gene mutations in cancers are shown, suggesting others are also candidate cancer genes needing to be tested. A C I N cancer cell-selective killing concept, utilizing synthetic lethality interactions between a C I N gene mutation and a second-site mutation, is discussed. Chapter 3 describes mutation testing o f the list o f candidate human C I N genes generated from Chapter 2 i n a panel o f colorectal cancer patients. The significance o f novel mutations, including genes involved i n sister chromatid cohesion is discussed with regards to the mutation frequency and prevalence o f mutations i n C L N tumors. Functional analysis o f several o f the mutations found i n colon cancers was performed i n yeast by introduction o f the mutations at the corresponding sites i n the yeast SMC1 gene and scoring the C I N phenotype. Chapter 4 describes the characterization o f 4 C L N genes identified i n the genomewide screens in Chapter 2. These 4 genes were only preliminarily characterized at the time the screens were completed. They potentially form an ubiquitin ligase complex. Phenotypic, genetic, and protein interaction data pertaining to this complex are analyzed and discussed. Chapter 5 draws conclusions from the above chapters, discusses the future directions o f research i n C I N , and show how yeast research can benefit cancer research.  30  T a b l e 1.1 G e r m l i n e mutations o f C I N and M I N genes causing cancer-prone  syndromes  Human gene  Yeast gene  Protein Function  Associated disease/syndrome  Major tumor types  MSH2,  MSH2,  MLHl,  MLHl  Mismatch repair (MMR)  Hereditary nonpolyposis colon cancer (HNPCC) (accounting for 3-5% of colorectal cancer)  Colon, uterus, endometrium, ovary  Base excision repair (BER) Nucleotide excision repair (NER) Double strand break (DSB) repair  MYH-associated polyposis (MAP) Xeroderma pigmentosum (XP)  Colon  Hereditary breast cancer  Breast, ovary  Nijmegen breakage syndrome (NBS) Ataxia Telangiectasia-like (ATL) Bloom Syndrome  Lymphoma, brain Myelodysplasia, acute myeloid leukemia Leukemia, lymphoma, skin  MSH6,  Mode of Inheritance Autosomal dominant  Reference  Autosomal recessive Autosomal recessive Autosomal dominant  (Al-Tassan et al., 2002) OMIM  (Fishel et al., 1993)  PMS2 MYH  mutY  /MUTYH  (E.coli)  XPA-G  RAD1-4, RAD14  BRCAl, BRCA2  Skin  NBS1  XRS2  MRE11A  MRE11  BLM  SGS1  Double strand break (DSB) repair Double strand break (DSB) repair D N A helicase  SGS1  D N A helicase  Werner syndrome  Bone, skin  Autosomal recessive  RECQL4  SGS1  D N A helicase  Rothmund-Thomson syndrome (RTS)  Bone, skin  Autosomal recessive  ATM  MEC1,  D N A damage checkpoint  Ataxia Telangiectasia (AT) Seckel syndrome  Leukemia, lymphoma, medulloblastomas and gliomas  Autosomal recessive  /RECQL3  WRN /RECQL2  TEL1  Autosomal recessive Autosomal recessive Autosomal recessive  (Tutt et al., 1999; Weaver et al., 2002) (Varon et al., 1998) (Stewart et al., 1999) (Mohaghegh and Hickson, 2001) (Mohaghegh and Hickson, 2001) (Mohaghegh and Hickson, 2001) (Savitsky et al., 1995)  Human gene  Yeast gene  P53, CHK2  RAD53  BUB1B  MAD3  APC  FANCA,B,C,D I,D2,E,F,G,I,J ,L,M  Protein Function  Associated disease/syndrome  Major tumor types  Mode of Inheritance  Reference  DNA damage checkpoint  Li-Fraumeni syndrome (LFS)  Autosomal dominant  (Varley et al., 1997) (Bell et al., 1999)  Mitotic spindle checkpoint Wnt signaling inhibition; chromosome segregation? repair of DNA interstrand crosslinks  Mosaic variegated aneuploidy (MVA) Familial adenomatous polyposis (FAP)  Soft tissue sarcomas and osteosarcomas, breast cancer, brain tumors, leukemia, and adrenocortical carcinoma Rhabdomyosarcoma, Wilms tumor, and leukemia Colon, thyroid, stomach, intestine  Autosomal recessive Autosomal dominant  (Hanks et al., 2004) (Green and Kaplan, 2003)  Fanconi anemia (FA)  Leukemia  Autosomal recessive & X-linked  (Niedernhof er et al., 2005)  T a b l e 1.2 K i n e t o c h o r e a n d s p i n d l e c h e c k p o i n t gene m u t a t i o n o r m i s r e g u l a t i o n associated w i t h cancer. (* shown as No. of positive patients or cell lines over the total No. tested) Gene  BUB1  BUB1B  Mutation/misregulation Dominant negative heterozygous deletion and missense mutation  Frequency* 2/19  Heterozygous missense mutation  1/30  Heterozygous missense mutation  1/10  Dominant negative heterozygous deletion in kinetochore localization domain  1/2  Deletion in kinetochore localization domain  2/2  Overexpressed  30/36  One heterozygous deletion, and one missense 2/19 mutation One heterozygous and one homozygous 3/10 missense mutation, one homozygous deletion  Tumor type  Reference (Cahill et al., Colorectal cancer 1998) (Gemma et Lung tumor al., 2000) Acute T-cell lymphoblastic (Ohshima et leukemia al., 2000) (Ru et al., Acute lymphoblastic leukemia 2002) (Ru etal., Hodgkin's lymphoma 2002) (Grabsch et Gastric cancer al., 2003) (Cahill et al., Colorectal cancer 1998) Acute T-cell lymphoblastic (Ohshima et leukemia al., 2000) (Shichiri et Colorectal cancer and others al., 2002) (Grabsch et Gastric cancer al., 2003) (Grabsch et Gastric cancer al., 2003) (Kim et al., Gastric cancer 2005) (Li and Breast cancer cell line Benezra, 1996) Nasopharyngeal cancer cell (Wang etal., lines 2000b) (Wang et al., Ovarian cancer cell lines 2002b) (Wang et al., Colorectal cancer 2004b) (Wang et al., Colorectal cancer 2004b) (Wang et al., Colorectal cancer 2004b) (Tomonaga Colorectal cancer et al., 2003) (Tomonaga Colorectal cancer et al., 2005) (de la Head and neck squamous eel Guardia et carcinomas al.,2001) (Ota etal., Salivary gland tumor 2002)  Downregulated (10 fold)  3/109  Overexpressed  19/28  Overexpressed  26/34  Missense mutation  22/49  Downregulated  1/1  Downregulated  2/5  Downregulated  3/7  Rod  Homozygous missense mutation  1/192  Zw10  Heterozygous missense mutation  2/192  Zwilch  Heterozygous premature truncation  1/192  CENP-A  Overexpressed (1.5-32.5 fold)  11/11  CENP-H  Overexpressed (1.7-9.6 fold)  15/15  Amplified (1.6-2.5 fold) Overexpressed (2.1-4.2 fold)  7/72 25/72  Overexpressed  25/26  Overexpressed  9/9  Cervical, acute lymphocytic leukemia, breast and colorectal cancer lines  Overexpressed  12/12  Thyroid cancer lines  Overexpressed  7/7  Colorectal cancer  Overexpressed (2.4-4.7 fold)  4/4  Colorectal cancer cell lines  BUB3  MAD2  CENP-F (mitosin) HEC1 (highly expressed in cancer; NDC80) Aurora-B (AIM1) INCENP  33  (Chen et al., 1997) (Sorrentino et al., 2005) (Tatsuka et al., 1998) (Adams et al.,2001)  F i g u r e 1.1 T h e b u d d i n g yeast c e l l c y c l e a n d c h r o m o s o m e c y c l e (adapted from P o t , 2 0 0 4 ) A. Yeast cells reproduce by budding. The cell cycle is divided into four stages [Gl, S (DNA replication), G2 and M (mitosis)]. The size of the bud gives an approximate indication of cell cycle stage. The nucleus is shown in red; in yeast, the nuclear membrane does not break down during mitosis. During cell division, chromosomes undergo a replication and segregation cycle that is synchronized with the cell cycle B. To follow the chromosome cycle in yeast, DNA content can be analyzed by flow cytometry of cells in which DNA has been stained with a fluorescent dye such as propidium iodide. A typical histogram showing the fluorescence distribution of a population of cycling cells is shown. Haploid cells in G l phase have a IN DNA content, while cells that have replicated their DNA and are undergoing mitosis (G2/M) have a 2N DNA content.  34  F i g u r e 1.2 O r g a n i z a t i o n o f centromere (reprinted from ( C l e v e l a n d et a l . , 2 0 0 3 ) C e n t r o m e r e s a n d k i n e t o c h o r e s : from epigenetics to m i t o t i c c h e c k p o i n t s i g n a l i n g , Cell, 1 1 2 , 4 0 7 - 4 2 1 , C o p y r i g h t 2 0 0 3 , w i t h p e r m i s s i o n from E l s e v i e r )  A. Overall organization of the centromere. A mitotic chromosome has been sectioned along the plane of the spindle axis, revealing the symmetric bipolar organization of a chromosome fully engaged on the spindle. (Right) Key elements have been pseudo colored. (Violet) The inner centromere, a heterochromatin domain that is a focus for cohesins and regulatory proteins such as Aurora B. (Red) The inner kinetochore, a region of distinctive chromatin composition attached to the primary constriction. (Yellow) The outer kinetochore, the site of microtubule binding, is comprised of a diverse group of microtubule motor proteins, regulatory kinases, microtubule binding proteins, and mitotic checkpoint proteins. B. Schematic illustration of centromere loci. Organization of centromeric DNA sequences from the four example organisms. (Top) Budding yeast with a 125 bp centromere comprised of three sequence domains (pink, red, yellow). Fission yeast centromeres show an organized structure, with a nonconserved central core (red), flanking inner repeats (pink arrows) at which the CENP-A-containing nucleosomes assemble, and conserved outer repeats (stippled purple). The Drosophila centromere spans -v.400 kb (red) embedded in constitutive heterochromatin (purple). (Bottom) Human centromeres have sizes approaching 10 Mb and are comprised of OJ-I satellite DNA (red) and a more divergent, less regular o i l satellite (pink), flanked by heterochromatin (purple).  35  F i g u r e 1 . 3 T h e process o f a c h i e v i n g b i p o l a r attachment (reprinted from ( P i n s k y a n d B i g g i n s , 2 0 0 5 ) T h e s p i n d l e c h e c k p o i n t : t e n s i o n versus attachment, Trends Cell Biol, 1 5 , 4 8 6 - 4 9 3 , C o p y r i g h t 2 0 0 5 , w i t h p e r m i s s i o n from E l s e v i e r ) Micro" utXites  Spindle pole  (a) Unattached kinetochores  Sister chromatid  (b) 'Search and Capture': Single kinetochore captured by the side of a probing spindle microtubule  (c) Sister chromatids transported to the pole where additional microtubules bind  (d) Sister kinetochore interacts with microtubule from the opposite pole  (e) Chromosomes congress to the spindle center and kinetochores come under tension TRENDS in Cell Boiogy  36  Figure 1.4 Types of kinetochore-microtubule attachments (reprinted from (Pinsky and Biggins, 2005) The spindle checkpoint: tension versus attachment, Trends Cell Biol, 15, 486-493, Copyright 2005, with permissionfromElsevier) (a) Amphitelic: either bipolar or bioriented attachment. Sister kinetochores face opposite poles and bind only microtubules arising from the adjacent pole. (b) Syntelic: sister kinetochores face the same pole and attach to microtubules emanating from that pole. (c) Monotelic: sister kinetochores face opposite poles but only one kinetochore binds microtubules, leaving an unattached kinetochore. (d) Merotelic: sister kinetochores face opposite poles but one (or both) kinetochore(s) interact with microtubules from both poles.  A m p h i t e l i c  Syntelic  Monotelic  TRENDS in Cell Biology  37  Structure of cohesin and a possible mechanism by which it might hold sister chromatids together (reprinted from (Nasmyth, 2002) Segregating sister genomes: the molecular biology of chromosome separation, Science, 2 9 7 , 559-565, Copyright 2002, with permission from AAAS) (A) Smcl (red) and Smc3 (blue) form intramolecular antiparallel coiled coils, which are organized by binge or junction domains (triangles). Smcl/3 heterodimers are formed through heterotypic interactions between the Smcl and Smc3 junction domains. The COOH terminus of Sec 1 (green) binds to Smcl's ABC-like ATPase head, whereas its NH terminus binds to Smc3's head, creating a closed ring. Scc3 (yellow) binds to Sccl's COOH-terminal half and does not make any direct stable contact with the Smcl/3 heterodimer. Sccl's separase cleavage sites are marked by arrows. Cleavage at either site is sufficient to destroy cohesion. By analogy with bacterial SMC proteins, it is expected that ATP binds both the Smcl and Smc3 heads, alters their conformation, and possibly brings them into close proximity. By altering Sccl's association with Smc heads, ATP binding and/or hydrolysis could have a role in opening and/or closing cohesin's ring. (B) Cohesin could hold sister DNA molecules together by trapping them both within the same ring. Cleavage of Sec 1 by separase would open the ring, destroy coentrapment of sister DNAs, and cause dissociation of cohesin from chromatin. F i g u r e 1.5  2  A  SMC3  Separase  3 8  F i g u r e 1.6 The stages o f mitosis (reprinted from (Weaver and Cleveland, 2005) Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death, Cancer Cell, 8, 7-12, Copyright 2005, with permission from Elsevier) Chromosomes enter mitosis as pairs of replicated sister chromatids that are linked by proteins known as cohesins. A: The chromatids condense during prophase and are released into the cytoplasm by nuclear envelope breakdown, which marks the transition into prometaphase and also represents the first irreversible transition into mitosis. B: During prometaphase, the initially unattached chromatids make connections to the microtubules of the mitotic spindle and the mitotic checkpoint is active, which means that the kinetochores assembled at the centromeres of unattached chromosomes generate a diffusible "wait anaphase" inhibitor. Antimitotic drugs delay cells in prometaphase by producing unattached kinetochores. C: At metaphase, every chromosome has made proper attachments to the mitotic spindle and has congressed to a central position. Production of the diffusible "wait anaphase" inhibitor has been silenced by stable kinetochore-microtubule interactions. As the checkpoint inhibitors decay, the anaphase promoting complex (APC), an E3 ubiquitin ligase, becomes active and recognizes securin and cyclin B, provoking their degradation. D: Loss of securin activates the protease, separase, that cleaves the cohesins, triggering sister chromatid separation and chromosome segregation during anaphase A. E: At anaphase B, the spindle elongates. F: At telophase, the now segregated chromosomes begin decondensing and the nuclear envelopes reform. G: Cytokinesis separates the nuclei into two daughter cells that re-enter interphase. g  PROMETAPHASE mitotic checkpoint  OH  METAPHASE mrtooc checkpoint OFF  ^ PROPHASE First stage ol mitosis  microtubule centrosome  ^  chromosome nuclear envelope INTERPHASE  O  checkpoint  *Ji2B  complexes  j-g  Cdc20 A C T I V A T I O N of A P C  g  C d e 2 0 activated Anaphase Promoting Complex  Ubiqutiration and degradation of securin  jbiq-Mir  chain  F  TELOPHASE final stage of mitosis  inactive separase active soparaso  E  o c  ANAPHASEB spindle elongation  39  ^ ANAPHASE A sister chromatid separation  F i g u r e 1.7 C e l l u l a r processes i n v o l v e d i n r e p l i c a t i o n and segregation o f c h r o m o s o m e s d u r i n g m i t o s i s (reprinted from ( L e n g a u e r et a l . , 1998) G e n e t i c i n s t a b i l i t i e s i n h u m a n cancers,  Nature, 3 9 6 , 6 4 3 - 6 4 9 , C o p y r i g h t 1998, w i t h p e r m i s s i o n from M a c m i l l a n  Publishers Ltd)  Processes involved include chromosome condensation, cohesion of sister chromatids, and centrosome/rnicrotubule formation and dynamics. Checkpoints that are required in chromosome replication and segregation include the mitotic spindle checkpoint, which ensures that chromosomes are aligned correctly before anaphase, and the DNA-damage checkpoint, which prevents cells with DNA damage from entering prophase. Aberrations in these processes and checkpoints could give rise to the CIN phenotype.  40  F i g u r e 1.8 M u l t i p l e roads to a n e u p l o i d y (reprinted f r o m ( R a j a g o p a l a n a n d L e n g a u e r , 2 0 0 4 a ) A n e u p l o i d y a n d cancer,  Nature,  432, 338-341, Copyright 2004, w i t h permission  from M a c m i l l a n Publishers Ltd)  The schematic illustrates a simplified cell cycle, highlighting processes that have been implicated in the advent of aneuploidy. Several pathways within the cell cycle (indicated in red) can be disrupted. Genes (indicated in green) associated with these processes and structures have been found to be mutated or functionally altered in aneuploid cancers.  G1/S checkpoint  41  F i g u r e 1.9 F l o w c h a r t o f d e v e l o p i n g therapeutic strategy b a s e d o n candidate C I N gene identification.  Identify C I N genes i n m o d e l o r g a n i s m s * S c r e e n for mutations i n candidate C I N genes i n t u m o r s Ar  F u n c t i o n a l studies o f c a n c e r gene mutants i n m o d e l o r g a n i s m s a n d h u m a n c e l l s A>  Synthetic lethal screen i n yeast u s i n g C I N m u t a t i o n D e t e r m i n e analogous s e c o n d a r y target i n h u m a n V a l i d a t e synthetic l e t h a l i t y i n h u m a n Ar  Determine pharmacological feasibility Ar  D r u g screen for s e c o n d a r y target  42  CHAPTER 2 Identification of Chromosome Instability Mutants in the Budding Yeast Saccharomyces cerevisiae and the Implication to Human Cancer  A modified version o f this chapter has been accepted for publication. Karen W . Y . Yuen*, Cheryl D . Warren*, O u Chen, Teresa K w o k , Phil Hieter, and Forrest A . Spencer (*These authors contributed equally to this work). Systematic Genome Instability Screens in Yeast and Their Potential Relevance to Cancer. Proceedings o f the National Academy o f Sciences o f the United States o f America.  43  2.1 I n t r o d u c t i o n  Genome instability is a hallmark o f cancer and falls into 2 classes: M I N (microsatellite instability, reflecting an increased mutation rate) or C I N (chromosome instability, reflecting an increased chromosome missegregation rate). While much is known about the spectrum o f germline and somatic mutations causing M I N i n cancer cells, little is known about the spectrum o f somatic gene mutations causing C I N in cancer cells (as described in Chapter 1). Recently, a cross-species candidate gene approach has been used to define - 2 0 % o f the C I N mutational spectrum i n colon cancer (Cahill et al., 1998; Rajagopalan and Lengauer, 2004b);(Wang et al., 2004b). To comprehensively identify additional C I N gene mutations i n cancers, one approach would be to identify all genes mutable to a C L N phenotype in a model organism, and to systematically test human homologues o f the model organism C I N gene set for somatic mutations i n tumors. Such a cross-species candidate gene approach, previously termed 'homologue probing' (Bassett et al., 1997), should in theory significantly expand our understanding o f the C L N mutational spectrum in cancer. Comprehensive identification o f genes whose mutation leads to C L N is an important, but daunting, goal yet to be achieved. Phenotype screening based on marker stability i n budding yeast by random mutagenesis has provided several gene collections (e.g. chromosome transmission fidelity (ctf), chromosome loss (chl), minichromosomes maintenance (mem), and chromosome instability (tin)), and these genes are often functionally conserved in other eukaryotes (Hoyt et al., 1990; Kouprina et al., 1988; Maine et al., 1984; Meeks-Wagner et a l , 1986; Ouspenski et al., 1999; Spencer et al., 1990). Not surprisingly, different genetic screens have led to identification o f gene sets important for various steps in the chromosome cycle, including those functioning at kinetochores, telomeres, and origins o f replication, or in microtubule dynamics, sister chromatid cohesion, D N A replication, D N A repair, D N A condensation and cell cycle checkpoints. A l l these processes must be executed at high fidelity and coordinated temporally and spatially within the cell cycle to maintain genetic integrity.  44  For instance, the chromosome transmission fidelity (ctf) mutant collection was generated by Spencer et al. (Spencer et al., 1990) i n the Hieter laboratory through mild E M S mutagenesis (0% killing, 10-fold increase i n canavanine resistant colonies), followed by screening o f -600,000 yeast colonies for an elevated colony sectoring phenotype, which reflects loss o f an artificial chromosome fragment. In total, 136 mutant strains were isolated. Based on complementation tests, this collection represents - 5 0 genes that could encode any o f the many components necessary for the chromosome cycle to proceed with high fidelity. Specific secondary screens have been applied to the ctf collection with the aim o f identifying mutants defective i n a particular structure or process (Doheny et al., 1993). To date, about half (24) o f the genes represented i n the ctf collection have been cloned and characterized (Table 2.1). A m o n g these, 9 genes encode kinetochore proteins, 10 encode proteins important for sister chromatid cohesion, and 5 encode other functions i n D N A / R N A metabolism. Both essential and non-essential genes were identified from the screen. Interestingly, the top 7 complementation groups altogether contain 75 alleles, representing over half o f the total number o f isolates. These 7 yeast genes are the most highly mutable to C I N in this particular assay. Despite the ease o f random mutagenesis and the possible recovery o f hypomorphs of essential genes, random mutagenesis approaches rarely achieve screen saturation, because mutability varies among genes due to differences i n size, base composition, and the frequency o f mutable sites that can lead to viable cells with a detectable phenotype. However, the use o f the S. cerevisiae gene knockout collection supports new and powerful strategies based on direct phenotyping o f the null mutants. The -4,700 nonessential gene-deletion mutants represent >70% o f yeast genes, but over 30% o f mutants remain functionally unclassified (Giaever et al., 2002; Winzeler et al., 1999) (Saccharomyces Genome Database, www.yeastgenome.org). In this study, I have used the gene knockout set to carry out 3 systematic screens that follow marker inheritance in different chromosomal contexts to identify genes important for maintaining genome stability i n yeast (i.e. non-essential yeast C I N genes). In addition to extending the catalog o f genes known to affect genome stability, several  45  themes emerge from the analysis o f the screen results. Because all mutants characterized are null, phenotype strength reflects the magnitude o f the role played b y each gene in genome stability. Thus direct comparisons are meaningful, between different mutants in a given assay system or between different assay systems for a given mutant. Some mutants exhibit phenotypes that are screen-specific, suggesting that chromosomal contexts determine what pathways predominate i n protecting against genomic change. Protein similarity was used to identify candidate C I N homologues i n other species, i n particular human genes with relevance to cancer. For yeast C I N genes whose human homologues are mutated i n cancers, yeast genetic interaction data were analyzed to identify common synthetic lethal interactors. Human homologues o f these common synthetic lethal interactors may be useful as drug targets with broad spectrum applicability for selective elimination o f C I N cancer cells.  46  2.2 M a t e r i a l s a n d m e t h o d s 2.2.1 G e n o m e - w i d e s c r e e n s  2.2.1.1 C T F s c r e e n The synthetic genetic array ( S G A ) selection scheme (Tong et al., 2001a) was used to introduce the ade2-101 ochre mutation and an artificial chromosome fragment (CF) into MATa deletion mutants obtained from Research Genetics (www.resgen.com). To construct donor strains used to mate with the MATa deletion mutant array, Y2454 (see Table 2.2 for strain list) was first co-transformed with 2 P C R products. One P C R product contained an ade2-101 allele and terminated with an adjoining 20-bp o f TEF promoter. The other P C R product contained the TEF promoter, the natMX cassette, the TEF terminator, and 40-bp overlap with genomic ADE2 downstream sequence. Transformants were selected on medium containing lOOmg/L c l o n N A T (Werner B i o Agents, Germany). Cointegration oiade2-l01 and natMX at the ADE2 gene locus was confirmed by P C R . The resulting strain (YPH1724) was crossed with either Y P H 2 5 5 or Y P H 1 1 2 4 , which contained C F V I I ( R A D 2 . d ) or C F I I I ( C E N 3 . L ) , respectively (Spencer et al., 1990). The 2 CFs were derived from different yeast chromosome arms and w i l l cover recessive mutations in that chromosomal region. The resulting diploids were sporulated, and spore progeny with the appropriate markers were recovered as the donor strains Y P H 1 7 2 5 and YPH1726. Two S G A analyses, each using one o f the donor strains, were performed as described previously (Tong et al., 2004) with the following modifications. The MATa yeast deletion mutant set (MATa ura3 his3 ykoA::kanMX) was arrayed at a density o f 768 colonies/omni tray for robotic pinning. Each deletion mutant strain was represented in duplicate i n each S G A analysis. The donor strain (MATa ade2-101::natMX ura3 his3 can! A mfalA::MFAlpr-HIS3),  containing a £/iL43-marked C F , was mated with yeast  deletion mutants on rich medium for 1 day. A l l plates were incubated at 2 5 ° C . Then diploids were selected twice (2 days and 1 day) on synthetic complete medium (SC) containing 200mg/L G418 (frivitrogen) and lacking uracil. Diploids were then pinned onto sporulation medium for 9 days, and MATa spore progeny with a C F were selected  47  on haploid selection medium (SC medium lacking histidine, uracil, and arginine but. containing 50mg/L-canavanine (Sigma)) for 2 days. Finally, MATa deletion mutants with a C F and the ade2-101 mutation were selected in 2 successive rounds (2 days each time) on the haploid selection medium with G418 and c l o n N A T . A l l strains from the final selection plate were streaked to single colonies on S C medium with 20% o f the standard adenine concentration. Plates were incubated at 25°C for 6-7 days, then at 4 ° C for 5-7 days to enhance the development o f red pigment. A n instability o f the C F was indicated by a colony color-sectoring phenotype as i n (Spencer et al., 1990). Briefly, red color in yeast cells is caused by accumulation o f pigment due to a block i n adenine production caused by the ade2-101 (ochre) mutation. This block is relieved i n the presence o f the SUP 11 gene located on the telocentric arm o f the C F , encoding an ochre-suppressing tRNA  T y r  . Cells that contain the C F are therefore unpigmented, whereas cells that do not  develop red color (Gerring et al., 1990; Hegemann et al., 1988; Warren et al., 2002). Colonies exhibiting unstable inheritance o f this C F develop red sectors, whereas w i l d type strains form mostly white colonies. The severity o f the phenotype was scored qualitatively by eye as mild, intermediate and severe (indicated as 1,2, and 3, respectively in Appendix 1 and Appendix 2). Figure 2.1.a shows the scheme o f the C T F screen, and examples o f severe and mild sectoring colonies. A l l deletion mutants that displayed a sectoring phenotype, or were identified in at least 1 o f the other screens ( B i M or A L F ) were retested for sectoring phenotype. A miniarray was constructed and S G A analyses were undertaken as described above. Deletion mutants showing a sectoring phenotype i n at least 2 out o f 8 isolates (either in the original genomic screen or in the retest) were scored as having a C T F phenotype.  2 . 2 . 1 . 2 Bimater  screen  The homozygous diploid deletion set obtained from Open Biosystems (www.openbiosystems.com) in 96-array format was grown on Y P D agar medium containing 200mg/L G418 for 3 days at 25°C. MATa and MATa mating tester lawns ( Y P H 3 1 5 and Y P H 3 1 6 respectively) were generated by spreading 2 m l o f saturated  48  culture on solid medium and grown at 25°C for 2 days. Each plate containing 96 deletion mutants was replica plated onto 4 test plates: an Y P D plate with 200mg/L G418 (a positive control), a synthetic complete medium plate lacking histidine, uracil, adenine, lysine, tryptophan and leucine (SC-6, a negative control), and 2 Y P D pre-mating plates. MATa and MATa mating tester lawns were each replica plated to the Y P D plate containing freshly replica plated deletion mutants. These were incubated at 25°C for 2 days for mating. The 2 Y P D plates were each replica plated to synthetic complete medium lacking histidine, uracil, lysine, adenine, tryptophan, and leucine (SC-6) and incubated at 25°C for 3 days to select mated products. These S C - 6 plates were visually inspected, and densitometry measurements were obtained for each deletion mutant using the QuantityOne program (BioRad). Deletion mutants that exhibited elevated mating rates with both MATa and MATa mating testers were identified as candidate bimaters for further study (Figure 2.1.b). For confirmation, all bimater candidates were retested for bimater phenotype along with all positive mutants from the other two screens ( A L F and C T F ) . For the retest, 4 independent isolates per mutant were patched in 1cm squares. Each plate also contained negative and positive control patches: wild-type diploid (YPH1738) and Ml A/Ml A, respectively. After selection, the'number o f colonies i n each patch was estimated. To minimize the effect o f early or late events during population growth, the median number o f colonies was used to calculate fold-change (mutant/wild-type ratio). Homozygous deletion mutants with an average o f > 1.5-fold increase in mating tests with both MATa and MATa testers were identified as bimaters. The severity o f each mutant phenotype was recorded as an estimate o f 2- to >5-fold increase over wild-type frequency after rounding (see Appendix 1 and 4).  2.2.1.3 a - l i k e f a k e r s c r e e n  The MATa haploid deletion collection (MATaykoA::kanMX)  obtained from  Research Genetics were manually arrayed in 1cm squares on Y P D plates. Each plate contained 3 controls: wild-type MATa (BY4742), wild-type MATa (BY4741), and the  49  MATa biml A:: kanMX from the MATa deletion collection. A lawn o f 5 x 10  MATahisl  mating tester cells (YPH316) freshly spread and dried onto solid rich medium. The presence o f a-type mating cells i n MATa mutant populations was detected as previously described (Warren et al., 2004a). ykoA patches were transferred onto the mating tester lawn by replica plating, followed by incubation at 30°C for 20-24 hr. Mated patches were then replica plated to SC-6, incubated for 2 days at 30°C to select mated products. Results from the primary screen were scored by comparing the number o f colonies per patch to the wild-type MATa control patch for that plate (Figure 2.1 .c). A l l positive mutants were retested as described above (along with all positive mutants from bimater and ctf screens), but using 4 independent isolates for each mutant. After selection, mated products were counted and a fold-change calculation was generated (mutant median colonies per patch/wild-type median colonies per patch). Mutants were scored as positive i f they exhibited > 2-fold increase over wild-type frequency.  2 . 2 . 2 Strain verification  To evaluate the mutant identity o f the 96-well content from each collection, cells from frozen stocks were patched on Y P D plates containing G418. A barely visible clump o f cells (~10 ) was added directly to 20 u l lysis buffer ( 2 5 m M T r i s - H C l pH8.0, 0.005% 5  sodium dodecyl sulfate, 14 m M (3-mercaptoethanol, 0.5 m M E D T A ) , and incubated at 85°C for 10 min. P C R was performed i n 96 well format using primers D I and K a n C for downtags (Shoemaker et al., 1996). P C R products were purified using Qiagen Qiaquick P C R purification kit or Macherey Nagel NucleoFast 96 P C R kit. D N A yield was determined using PicoGreen (Molecular Probes), and the concentration o f each well was adjusted to 10-50 ng/ml. The downtag sequence was determined using oligonucleotide 5'-catctgcccagatgcgaagttaag-3' as primer. For mutants lacking a downtag, or when the downtag sequence analysis was ambiguous, the uptag sequence was obtained from P C R products generated using primers U l and K a n B , followed by sequencing obtained using a K a n B l (Shoemaker et al., 1996) sequencing primer.  50  T h e sequence o f the tag P C R products w a s d e t e r m i n e d b y d i d e o x y t e r m i n a t i o n at the J H M I S e q u e n c i n g C o r e F a c i l i t y o r S e q W r i g h t Incorporated ( w w w . s e q w r i g h t . c o m ) . T h e results w e r e a n a l y z e d u s i n g B L A S T N against a tag database, a n d a l i g n m e n t s w i t h expected tags h a v i n g e-values < 1 0 "  10  were considered evidence o f w e l l validation.  S e q u e n c e traces for B L A S T n outcomes > 1 0 "  10  w e r e read m a n u a l l y . A m o n g these w e r e  c l e a n traces i n d i c a t i n g tag mutations present i n yeast that prevented a l i g n m e n t w i t h the correct tag sequences w i t h significant e-value ( E a s o n et a l . , 2 0 0 4 ) , o r traces w i t h t w o o r m o r e peaks at m a n y p o s i t i o n s . O f t e n w h e n t w o tags w e r e present, the i d e n t i t y o f a c o n t a m i n a t i n g mutant i n a g i v e n w e l l c o u l d b e determined. I n h o m o z y g o u s d i p l o i d s , different b u t ' c o r r e c t ' tag alleles w e r e often noted (e.g. w h e r e o n e tag w a s a  frameshifted  v e r s i o n o f the expected sequence) ( E a s o n et a l . , 2 0 0 4 ) . F a i l u r e i n mutant v e r i f i c a t i o n b y tag s e q u e n c i n g is c l a s s i f i e d as " w r o n g " (incorrect strain(s) present), " c o n t a m i n a t i o n " (correct strain present b u t a c o n t a m i n a t i n g strain w a s evident), o r " n d " (not d e t e r m i n e d because the sequence o b t a i n e d w a s unreadable, o r that d e l e t i o n c o l l e c t i o n c o n t a i n e d n o yeast to validate). Subsets o f C L N mutants w e r e freshly generated b y transformation a n d p h e n o t y p e d . T h e y k o A : .kanMXcassette  w a s P C R from the d e l e t i o n set mutant w i t h gene  f l a n k i n g p r i m e r s l o c a t e d ~ 3 0 0 b p upstream a n d d o w n s t r e a m o f the gene, a n d the P C R p r o d u c t w a s transformed i n the respective parental w i l d - t y p e . T r a n s f o r m a n t s w e r e c o n f i r m e d b y p r i m e r s f l a n k i n g the P C R p r o d u c t to c o n f i r m integration o f the d e l e t i o n a l l e l e a n d ensure r e m o v a l o f the w i l d - t y p e l o c u s .  2.2.3 Bioinformatic analysis 2.2.3.1 Functional analysis Over-representation o f G O b i o l o g i c a l process a n d c e l l u l a r c o m p o n e n t annotation i n the yeast C I N gene list, c o m p a r e d to a l l yeast genes, w a s d e t e r m i n e d u s i n g G O T e r m F i n d e r as o f M a y 3, 2 0 0 6 ( d b . y e a s t g e n o m e . o r g / c g i - b i n / G O / g o T e r m F i n d e r . p l ) .  51  2.2.3.2 BLAST analysis P r o t e i n sequences o f yeast C I N genes w e r e u s e d as queries i n a B L A S T p a l i g n m e n t search against p r o t e i n sequence d o w n l o a d s for Homo sapiens ( R e f S e q p r o t e i n database, as o f J u n e 2 0 0 4 ) , Mus musculus ( R e f S e q p r o t e i n database June 2 0 0 4 ) , C . elegans ( W o r m b a s e June 2 0 0 4 ) , D. melanogaster ( F l y B a s e release 3.2.0), a n d S. pombe (Sanger Institute, p o m p e p June 2 0 0 4 ) . H u m a n proteins f r o m R e f S e q w i t h B L A S T p a l i g n m e n t s to yeast C I N p r o t e i n queries (e-value <10" , J u l y 2 0 0 4 ) w e r e searched against 10  O M I M ( w w w . n c b i . n l m . n i h . g o v / o m i m ) a n d cancer census ( F u t r e a l et a l . , 2 0 0 4 ) p r o t e i n datasets for disease, e s p e c i a l l y cancer, a s s o c i a t i o n .  2.2.3.3 Protein and synthetic lethal interaction network P r o t e i n - p r o t e i n interactions a n d genetic interactions w i t h yeast C I N genes w e r e o b t a i n e d f r o m the G R I D database, a n d the i n t e r a c t i o n n e t w o r k s w e r e v i s u a l i z e d t h r o u g h the O S P R E Y p r o g r a m ( v l . 2 . 0 ) ( B r e i t k r e u t z et a l . , 2 0 0 3 a ; B r e i t k r e u t z et a l . , 2 0 0 3 b ) .  2.2.4 Electrophoretic karyotype of a-like fakers F o r the electrophoretic k a r y o t y p e a n a l y s i s , a MATa his5A::kanMX tester strain w a s used. T h i s strain contains a c h r o m o s o m e III l e n g t h p o l y m o r p h i s m that d i s t i n g u i s h e s it from c h r o m o s o m e III o f the d e l e t i o n c o l l e c t i o n b a c k g r o u n d . M a t i n g b e t w e e n  MATa  strains w i l l o c c u r w h e n the MATa l o c u s from either parental genotype i s lost. D u r i n g the A L F screen, the b a s a l rate o f loss for the MATa hisl m a t i n g tester ( Y P H 3 1 5 ) w a s o b s e r v e d to b e m u c h l o w e r than that o f B Y 4 7 4 2 (parental strain to the MATa d e l e t i o n c o l l e c t i o n ) . T h u s , n e a r l y a l l events detected w e r e d u e to g e n o m e i n s t a b i l i t y i n the d e l e t i o n mutant b e i n g characterized. S a m p l e preparation, p u l s e d f i e l d g e l a n a l y s i s , a n d i n - g e l h y b r i d i z a t i o n s w e r e p e r f o r m e d as d e s c r i b e d i n ( W a r r e n et a l . , 2 0 0 4 a ) .  52  2.3 Results 2.3.1 Genome-wide marker loss screens identify 130 yeast deletion mutants T h r e e c o m p l e m e n t a r y m a r k e r loss assays w e r e p e r f o r m e d u s i n g the n o n - e s s e n t i a l g e n e - d e l e t i o n mutant set. In the first screen ( C T F , for c h r o m o s o m e t r a n s m i s s i o n f i d e l i t y , F i g u r e 2.2a), inheritance o f an a r t i f i c i a l c h r o m o s o m e fragment ( C F ) w a s m o n i t o r e d u s i n g a c o l o n y c o l o r m a r k e r . I m o d i f i e d the S y n t h e t i c G e n e t i c A r r a y ( S G A ) m e t h o d o l o g y ( T o n g et a l . , 2 0 0 1 a ) to construct h a p l o i d d e l e t i o n strains c a r r y i n g a C F , a n d p e r f o r m e d a c o l o n y c o l o r - s e c t o r i n g assay as a n i n d i c a t o r o f c h r o m o s o m e i n s t a b i l i t y ( H i e t e r et a l . , 1985; S p e n c e r et a l . , 1990). L i n e a r a r t i f i c i a l C F s serve as sensitive i n d i c a t o r s because their presence o r absence does not affect v i a b i l i t y , a n d they r e s e m b l e n a t u r a l c h r o m o s o m e s i n their structure and s t a b i l i t y ( w i t h 1.7-7 loss events per 1 0 d i v i s i o n s ) 4  ( G e r r i n g et a l , 1990; H e g e m a n n et a l . , 1988; K o s h l a n d and H i e t e r , 1987; S h e r o et a l . , 1 9 9 1 ; W a r r e n et a l . , 2 0 0 2 ) . S i n c e the m a r k e r s o n the C F are s u r r o u n d e d b y sequences that h a v e n o s i m i l a r i t y to the yeast g e n o m e , loss o f m a r k e r s p r i m a r i l y represent loss o f the whole C F . In the s e c o n d and t h i r d screens, an endogenous l o c u s (the m a t i n g type l o c u s MAT o n c h r o m o s o m e III) w a s e x p l o i t e d as a m a r k e r . A b i m a t e r screen (designated B i M , F i g u r e 2.2.b) f o l l o w e d inheritance o f the  MATa a n d MATaloci i n h o m o z y g o u s d i p l o i d  d e l e t i o n mutants. D i p l o i d c e l l s h e t e r o z y g o u s at MAT d o not mate due to c o d o m i n a n t s u p p r e s s i o n o f h a p l o i d - s p e c i f i c c e l l differentiation p a t h w a y s . L o s s o f either the the  MATa o r  MATa a l l e l e results i n m a t i n g c o m p e t e n c e , w h e r e the m a t i n g type i s d e t e r m i n e d b y  the r e m a i n i n g allele. R e c i p r o c a l m a t i n g tests w i t h M 4 7 a o r  MATa m a t i n g testers w e r e  p e r f o r m e d o n the h o m o z y g o u s d i p l o i d d e l e t i o n set to i d e n t i f y c e l l p o p u l a t i o n s w h i c h form mated products w i t h both  MATa a n d MATa at h i g h rates. T h e endogenous rate o f  loss o f either MAT a l l e l e i n w i l d - t y p e c e l l s i s 2-4 events i n 1 0 d i v i s i o n s ( L i r a s et a l . , 5  1978; S p e n c e r et a l . , 1990), w h e r e the p r e d o m i n a n t m e c h a n i s m i s m i t o t i c r e c o m b i n a t i o n b e t w e e n h o m o l o g u e s . T h i s loss o f h e t e r o z y g o s i t y c a n also be due to c h r o m o s o m e l o s s , c h r o m o s o m a l rearrangement (deletions or translocations w i t h loss), o r gene c o n v e r s i o n (allele replacement).  53  I n the t h i r d screen (designated A L F , for a - l i k e faker, F i g u r e 2.2.c), the MATa l o c u s inheritance w a s s i m i l a r l y f o l l o w e d i n the MATa h a p l o i d d e l e t i o n set b y a m a t i n g test. T h e MATa l o c u s encodes t r a n s c r i p t i o n factors that suppress a-specific a n d p r o m o t e a - s p e c i f i c gene e x p r e s s i o n (Strathern et a l . , 1981). L o s s o f the MATa l o c u s leads to the default m a t i n g type i n yeast, w h i c h is the a-type differentiation state. T h u s , MATa c e l l s that lose the MAriocus  w i l l mate as a-type c e l l s , a n d are c a l l e d ' a - l i k e fakers' (Strathern  et a l . , 1981). T h e frequency o f a - l i k e faker c e l l s i n a p o p u l a t i o n i s detected b y p r o t o t r o p h i c s e l e c t i o n o f m a t e d products. I n w i l d - t y p e yeast, A L F m i t o t i c segregants are generated at a rate o f ~ 1 0 " ( ( H e r s k o w i t z , 1988b); C D W a n d F A S u n p u b l i s h e d ) . 6  M e c h a n i s m s l e a d i n g to MATa l o c u s loss i n MATa c e l l s are s i m i l a r to L O H i n d i p l o i d c e l l s , except that i n h a p l o i d s there is n o h o m o l o g f o r m i t o t i c r e c o m b i n a t i o n . H o w e v e r , the silent m a t i n g type l o c u s HMRa c a n m i t o t i c a l l y r e c o m b i n e w i t h the MATa l o c u s (see below). T h e mutants i d e n t i f i e d i n the 3 assays w e r e subjected to a d d i t i o n a l v a l i d a t i o n s . F i r s t , mutants from each p r i m a r y screen w e r e retested b y a l l 3 assays to ensure p h e n o t y p e r e p r o d u c i b i l i t y . 3 1 0 k n o c k o u t strains w e r e i d e n t i f i e d after secondary s c r e e n i n g ( 8 4 C T F , 130 B i M , a n d 2 4 7 A L F ) . N e x t , the effect o f c r o s s - w e l l c o n t a m i n a t i o n w a s evaluated b y d e t e r m i n i n g the i d e n t i t y o f the d e l e t i o n mutations present i n each o f the 3 1 0 w e l l l o c a t i o n s i n each o f the 3 d e l e t i o n arrays (see A p p e n d i x 1 a n d 2 f o r details). T h i s w a s a c c o m p l i s h e d b y s e q u e n c i n g the o l i g o n u c l e o t i d e ' t a g ' u n i q u e to each d e l e t i o n a l l e l e ( G i a e v e r et a l . , 2 0 0 2 ) . T h e presence o f >1 tag sequence o r a n incorrect t a g sequence w a s e v i d e n c e o f c o n t a m i n a t i n g o r w r o n g d e l e t i o n strains, a n d the p h e n o t y p e s o f these l o c a t i o n s w e r e d i s c a r d e d . T h e 3 1 0 w e l l p o s i t i o n s e x h i b i t e d 2 2 % , 9 % , a n d 1 4 % error i n the MATa, MATa, a n d h o m o z y g o u s d i p l o i d sets, r e s p e c t i v e l y . A f t e r adjustment, 2 9 3 k n o c k o u t strains w e r e v e r i f i e d as e x h i b i t i n g C I N i n at least 1 o f the 3 assays. O f these, 2 1 0 ( 7 2 % ) w e r e u n c o n t a m i n a t e d i n a l l 3 sets. T o investigate the o v e r a l l error rate i n e a c h d e l e t i o n set, w e sequenced strains from 6 0 r a n d o m l y c h o s e n w e l l addresses a n d f o u n d 1 2 % , 3 % , a n d 3 % c o n t a m i n a t e d w e l l s i n MATa, MATa, a n d h o m o z y g o u s d i p l o i d sets, r e s p e c t i v e l y . T h e h i g h e r error rate a m o n g yeast C I N mutants, relative to a r a n d o m l y  54  c h o s e n set, m a y reflect a h i g h e r representation o f s l o w - g r o w i n g yeast strains a m o n g the C L N gene set that are r e a d i l y r e p l a c e d b y faster g r o w i n g c o n t a m i n a n t s . T h e s e tag sequence analyses suggest that the false negative frequency w a s b e t w e e n 3 a n d 2 2 % (i.e. p h e n o t y p e d e t e c t i o n cannot be p e r f o r m e d due to the absence o r c o n t a m i n a t i o n o f m u t a n t from the appropriate p o s i t i o n i n a c o l l e c t i o n ) . S e v e r a l different error o r i g i n s w e r e observed i n each collection. These included neighboring-well spillover (20-50% o f cases), plate-to-plate c a r r y o v e r (mutants from a c o n s e r v e d w e l l p o s i t i o n but from a different plate, 5 - 1 5 % o f cases), c o m m o n substitution b y a s i n g l e recurrent strain (that c o u l d o c c u r from m e d i a c o n t a m i n a t i o n , ~ 1 0 % ) , a n d i n d i v i d u a l events w i t h n o apparent p h y s i c a l pattern ( 3 0 - 5 0 % o f cases). F i n a l l y , a n a d d i t i o n a l source o f artifact i n d e l e t i o n c o l l e c t i o n p h e n o t y p i n g i s the o c c a s i o n a l presence o f u n d e s i r e d ' s e c o n d a r y ' m u t a t i o n s that cause the p h e n o t y p e b e i n g screened (i.e. p o s i t i v e phenotypes c a u s e d b y m u t a t i o n s that are not at the site o f the k n o c k o u t allele). G a i e v e r et al. estimated the presence o f lethal o r s l o w - g r o w t h p h e n o t y p e s c a u s e d b y m u t a t i o n s i n genes that d o not segregate w i t h a k n o c k o u t a l l e l e to o c c u r at a frequency o f 6 . 5 % ( G i a e v e r et a l . , 2 0 0 2 ) . S u c h " c o l l a t e r a l d a m a g e " w o u l d be e x p e c t e d to o c c u r at an e v e n h i g h e r frequency i n non-essential genes. T o v e r i f y that the C I N p h e n o t y p e is a c t u a l l y due to the k n o c k o u t a l l e l e , subsets o f mutants w e r e regenerated b y independent t r a n s f o r m a t i o n a n d p h e n o t y p e d . M u t a n t s w i t h p h e n o t y p e s i n at least 2 screens w e r e r e c o n f i r m e d as C I N mutants i n n e w transformants at a h i g h rate (13/13 C T F , 9/10 B i M , 9/11 A L F ) . O n the other h a n d , mutants i d e n t i f i e d w i t h p h e n o t y p e s i n o n l y a s i n g l e assay w e r e r e c o n f i r m e d i n n e w transformants at l o w e r rates, - 4 3 % for the h a p l o i d c o l l e c t i o n s (2/6 o f mutants e x h i b i t i n g C T F o n l y , 4/8 o f mutants w i t h A L F p h e n o t y p e o n l y , a n d - 7 5 % for the d i p l o i d c o l l e c t i o n (3/4 o f mutants e x h i b i t i n g B i M p h e n o t y p e o n l y ) . T h e s e data i n d i c a t e a s i g n i f i c a n t frequency o f s e c o n d a r y m u t a t i o n effects i n the a s s a y - s p e c i f i c subsets o f C I N mutants i d e n t i f i e d i n the p r i m a r y screens, a n d e m p h a s i z e the v a l i d a t i o n inherent i n p e r f o r m i n g screens i n m u l t i p l e c o l l e c t i o n s . T h e h i g h e r r e c o n f i r m a t i o n rate o f B i M from the h o m o z y g o u s d e l e t i o n mutants i s consistent w i t h the presence o f s e c o n d a r y m u t a t i o n s , w h i c h w o u l d often be c o v e r e d b y the w i l d - t y p e  55  a l l e l e d u r i n g the c o n s t r u c t i o n o f d i p l o i d s w h e n independent h a p l o i d segregants w e r e mated. In total, 130 mutants are o f h i g h c o n f i d e n c e (the 115 d e l e t i o n strains i d e n t i f i e d i n m o r e than 1 assay, together w i t h 15 mutants r e c o n f i r m e d i n d e p e n d e n t l y to h a v e a p o s i t i v e p h e n o t y p e i n o n l y 1 assay). T h e s e 130 genes are l i s t e d i n F i g u r e 2.3 a n d A p p e n d i x 1, w h i c h reflect the current data status i n c l u d i n g a l l c o n f i r m a t i o n s p e r f o r m e d to date. T h e r e m a i n i n g 163 mutants i d e n t i f i e d i n o n l y 1 screen are l i s t e d i n A p p e n d i x 2 , a n d are regarded w i t h l o w e r c o n f i d e n c e ( w i t h - 4 3 % a n d - 7 5 % true p o s i t i v e frequencies a m o n g the a s s a y - s p e c i f i c subsets i d e n t i f i e d i n the h a p l o i d and d i p l o i d mutant screens, r e s p e c t i v e l y ) . A p p e n d i x 1 a n d 2 i n c l u d e measures o f p h e n o t y p e severity, as w e l l as annotations for w e l l c o n t a m i n a t i o n i n a n y o f the 3 d e l e t i o n sets a n d changes d u e to independent k n o c k o u t e v a l u a t i o n .  2.3.2 Functional distribution of yeast CIN genes C o m p a r i n g the gene o n t o l o g y ( G O ) annotations o f the 293 C L N genes to that o f the entire yeast g e n o m e ( H a r r i s et a l . , 2 0 0 4 ) i n d i c a t e d that the C I N gene set has a n o v e r representation i n n u m e r o u s expected c e l l u l a r c o m p o n e n t s : n u c l e u s , c h r o m o s o m e , k i n e t o c h o r e , m i c r o t u b u l e , c y t o s k e l e t o n , s p i n d l e , n u c l e a r pore, s p i n d l e p o l e b o d y , r e p l i c a t i o n fork, and c h r o m a t i n ( A p p e n d i x 3). F o r G O b i o l o g i c a l processes, the C L N gene list i s e n r i c h e d i n genes i n v o l v e d i n c e l l c y c l e , c e l l p r o l i f e r a t i o n , response to D N A d a m a g e response, and n u c l e a r d i v i s i o n ( A p p e n d i x 4 ) . U s i n g G O b i o l o g i c a l process annotation, the 2 9 3 C I N genes f a l l i n b r o a d f u n c t i o n a l groups, i n c l u d i n g - 4 0 % functioning i n D N A metabolism, chromosome, or cell cycle, - 4 0 % functioning i n processes n o t o b v i o u s l y i m p l i c a t e d i n m a r k e r loss, and - 2 0 % w i t h u n k n o w n f u n c t i o n ( F i g u r e 2.4). T h e s e G O annotations reflect the current k n o w l e d g e o f studied genes, i n d i c a t i n g that these screens i d e n t i f i e d genes k n o w n to b e f u n c t i o n i n g i n g e n o m e maintenance. Interestingly, genes not p r e v i o u s l y k n o w n to contribute to s t a b i l i t y w e r e also i d e n t i f i e d . F o r e x a m p l e , 7 yeast mutants i n the adenine b i o s y n t h e t i c p a t h w a y {adel, ade2, ade4, ade5/7, ade6, ade8, adel 7) gave rise to elevated a - l i k e fakers at frequencies  56  r a n g i n g from 2 - to 3 1 - f o l d a b o v e w i l d - t y p e ( A p p e n d i x 1 and 4). T h r e e o f these (adel, ade6 a n d adel 7, s h o w n i n F i g u r e 2.3) w e r e tested i n fresh transformants, a n d a l l 3 w e r e v a l i d a t e d . T h i s indicates that adenine, adenine p a t h w a y intermediates o r d e r i v a t i v e metabolites are i m p o r t a n t for g e n o m e stability, a n d that c o m p e n s a t o r y m e c h a n i s m s u s e d b y c e l l s w h e n de n o v o adenine synthesis i s b l o c k e d are not f u l l y sufficient. U s i n g the G R I D database and O S P R E Y n e t w o r k v i s u a l i z a t i o n p r o g r a m , 9 2 p h y s i c a l interactions w e r e f o u n d a m o n g 103 o f 2 9 3 C I N proteins, i n c l u d i n g p r o t e i n c o m p l e x e s , n e t w o r k s and p a t h w a y s that are k n o w n to be important for m a i n t a i n i n g c h r o m o s o m e s t a b i l i t y ( B r e i t k r e u t z et a l . , 2 0 0 3 a ; B r e i t k r e u t z et a l . , 2 0 0 3 b ) ( F i g . 2.7). I n s p e c t i o n o f n e t w o r k interactions s h o u l d r e v e a l n o v e l hypotheses r e g a r d i n g functions o f u n c h a r a c t e r i z e d genes. F o r e x a m p l e , msb2A w a s i d e n t i f i e d as a bimater, a n d M s b 2 p p h y s i c a l l y interacts w i t h M a d 2 p a n d M a d 3 p i n the s p i n d l e c h e c k p o i n t p a t h w a y a n d N d c 8 0 p at the k i n e t o c h o r e . W h i l e M s b 2 p is k n o w n to be a n o s m o s e n s o r p r o t e i n a n d is r e q u i r e d to establish c e l l p o l a r i t y , its r o l e i n m a i n t a i n i n g c h r o m o s o m e s t a b i l i t y has not b e e n e x p l o r e d . YBP2, i d e n t i f i e d i n the b i m a t e r test, is i m p l i c a t e d i n o x i d a t i v e stress response a n d the gene p r o d u c t interacts w i t h N u p l 4 5 p , w h i c h i s essential. N u p l 4 5 p interacts w i t h N u p l 2 0 p a n d N u p 8 4 p , w h i c h w e r e f o u n d i n o u r screens a l o n g w i t h another n u c l e o p o r i n N u p l 3 3 p , suggesting that n u c l e o p o r i n s p l a y i m p o r t a n t roles i n m a i n t a i n i n g c h r o m o s o m e stability. A s u b c o m p l e x o f n u c l e o p o r i n s c o n t a i n i n g N u p 5 3 p , N u p l 7 0 p , a n d N u p l 5 7 p are associated w i t h the s p i n d l e c h e c k p o i n t proteins M a d l p a n d M a d 2 p . Interestingly, ybp2A is synthetic l e t h a l w i t h mad2A, suggesting Y b p 2 p m a y p l a y a r o l e i n g e n o m e s t a b i l i t y i n a s s o c i a t i o n w i t h n u c l e o p o r i n s a n d the s p i n d l e c h e c k p o i n t . Indeed, Y b p 2 p w a s r e c e n t l y f o u n d to interact w i t h m u l t i p l e k i n e t o c h o r e proteins, a n d w a s f o u n d to s p e c i f i c a l l y interact w i t h centromere D N A sequences b y c h r o m a t i n immunoprecipitation (Kentaro O h k u n i and K a t s u m i K i t a g a w a , personal communication). I n a d d i t i o n , several C I N genes i d e n t i f i e d i n the screens w e r e r e c e n t l y c h a r a c t e r i z e d to p l a y a r o l e i n g e n o m i c stability. F o r e x a m p l e , D i a 2 p , a F - b o x p r o t e i n i n the S C F ( S k p l p C d c 5 3 p / C u l l i n - F - b o x ) E 3 u b i q u i t i n ligase c o m p l e x , w a s r e c e n t l y s h o w n to b e i n v o l v e d i n r e g u l a t i n g D N A r e p l i c a t i o n a n d important for stable passage o f r e p l i c a t i o n forks t h r o u g h  57  r e g i o n s o f d a m a g e d D N A a n d natural fragile r e g i o n s ( B l a k e et a l . , 2 0 0 6 ; K o e p p et a l . , 2 0 0 6 ) . N c e 4 p is i n v o l v e d i n m e d i a t i n g S g s l p - T o p 3 p h e l i c a s e - t o p o i s o m e r a s e c o m p l e x ( C h a n g et a l . , 2 0 0 5 ; M u l l e n et a l . , 2 0 0 5 ) , and M m s 2 2 p a n d M m s l p are i n a n o v e l D N A d a m a g e r e p a i r p a t h w a y ( B a l d w i n et a l . , 2 0 0 5 ) (see C h a p t e r 4 ) . Integrating the C L N gene c a t a l o g w i t h other p h e n o t y p i c , genetic and p h y s i c a l i n t e r a c t i o n data p r o v e s to b e a fruitful avenue to further o u r u n d e r s t a n d i n g o f m e c h a n i s m s that m a i n t a i n g e n o m i c stability.  2.3.3 Integration of genome-wide phenotypic screen with genetic screens reveals functions of uncharacterized genes in chromosome stability maintenance U s i n g 14 h y p o m o r p h i c a n d 3 h y p e r m o r p h i c k i n e t o c h o r e alleles as queries, g e n o m e - w i d e synthetic l e t h a l ( S L ) a n d synthetic dosage l e t h a l i t y ( S D L ) screens w e r e p e r f o r m e d o n the non-essential yeast d e l e t i o n mutant set ( M e a s d a y et a l . , 2 0 0 5 ) . S L interactions o c c u r b e t w e e n genes i n v o l v e d i n the same, p a r a l l e l or redundant b i o l o g i c a l pathway. S D L interaction occurs w h e n overexpression o f a protein i n wild-type cells r e m a i n s v i a b l e , but causes l e t h a l i t y i n a mutant. S D L c a n o c c u r b e t w e e n genes w i t h i n the same c o m p l e x w h e r e their s t o i c h i o m e t r y i s important. O v e r e x p r e s s i o n o f the q u e r y p r o t e i n m a y titrate out another p r o t e i n that is r e q u i r e d for c e l l v i a b i l i t y i n the k n o c k o u t strain. I f the f u n c t i o n o f the q u e r y p r o t e i n i s to regulate the mutant p r o t e i n , then o v e r e x p r e s s i o n o f the r e g u l a t o r y factor c o u l d be detrimental to the k n o c k o u t mutant. C o n v e r s e l y , i f the n o r m a l f u n c t i o n o f the mutant p r o t e i n is to regulate the q u e r y p r o t e i n , then o v e r e x p r e s s i n g the q u e r y p r o t e i n i n a strain defective for its r e g u l a t o r y factor c o u l d b e l e t h a l ( M e a s d a y a n d H i e t e r , 2 0 0 2 ) . T h e k i n e t o c h o r e S L a n d S D L screens i d e n t i f i e d 211 non-essential d e l e t i o n mutants i n total, but s u r p r i s i n g l y , o n l y 14 gene mutants w e r e i d e n t i f i e d i n b o t h S L a n d S D L screens. H o w e v e r , these o v e r l a p p i n g mutants w e r e e n r i c h e d for c h r o m o s o m e t r a n s m i s s i o n f i d e l i t y (ctf) defects (8/14 mutants from b o t h S L a n d S D L screens v s . 2 0 / 1 9 7 mutants from either S L or S D L screens d i s p l a y e d a ctf p h e n o t y p e ) ( r e v i e w e d i n ( B a e t z et a l . , 2 0 0 6 ; E i s e n s t e i n , 2005)). O n e gene i d e n t i f i e d i n this o v e r l a p p i n g set and the ctf screen w a s RCS1IAFT1,  an iron-regulated transcription  factor ( R u t h e r f o r d a n d B i r d , 2 0 0 4 ) . Indeed, R c s l p c o - l o c a l i z e s w i t h a k i n e t o c h o r e p r o t e i n  58  by indirect immunofluorescence analysis on chromosome spreads, and has both genetic and physical interactions with the inner kinetochore protein Cbflp (Measday et al., 2005). Such an example illustrates the power of complementing genome-wide SL, S D L and phenotypic screens to uncover hidden relationships and predict functions of genes.  2.3.4 Chromosome loss is the major mechanism of M47a loss in a-like fakers The CTF and B i M phenotypes have been widely used to study genome instability. However, the A L F phenotype has been only rarely used (Lemoine et al., 2005; Liras et al., 1978; Warren et al., 2004a), and has not been as well characterized. The electrophoretic karyotype of mated colonies obtained after selection was analyzed to infer the mechanism of MATa locus loss. Possible events include loss of the entire chromosome III, deletion or translocation removing the MAT locus, or gene conversion from MATa to MATa by recombination with HMRa. The chromosome III in the mating tester strain was larger than that in the knockout strains, and could be visually differentiated by pulsed-field gel electrophoresis (Figure 2.6a). In-gel hybridizations with a probe that hybridizes to 3 distant sites on chromosome III (the MAT locus, and the silent mating type loci located distally on each arm) allowed detection of the 2 parental chromosome III bands as well as aberrant chromosome III derivatives. Aberrant chromosomes III were observed in a variety of sizes, including a 200 kb product likely to represent homologous recombination between MATa  and the silent locus HMRa, known  to generate an active MATa locus concomitant with a large deletion on the right arm of chromosome III (reviewed in (Herskowitz, 1988a)). Electrophoretic karyotyping of mated products from wild-type indicated that 68% of events were due to whole chromosome loss, 20% to chromosomal rearrangement, and 12%> to gene conversion (Figure 2.6b). Karyotype analysis of 13 highfrequencyA L F mutants showed that in 11 A L F mutants, loss of whole chromosome III was the predominant mechanism, similar to wild-type cells. In 2 A L F mutants, different predominant mechanisms were observed in a statistically significant manner. rad27A showed predominantly chromosome rearrangement, whereas most sovlA had an intact  59  c h r o m o s o m e III. RAD27 encodes a n endonuclease that p r o m o t e s O k a z a k i fragment m a t u r a t i o n d u r i n g D N A r e p l i c a t i o n . T h e A L F associated rearrangements are consistent w i t h p r e v i o u s c h a r a c t e r i z a t i o n o f RAD2'7 as a gene that protects against gross c h r o m o s o m a l rearrangements ( C h e n a n d K o l o d n e r , 1999). SO VI has b e e n i m p l i c a t e d i n r e s p i r a t i o n b a s e d o n its l o c a l i z a t i o n to the m i t o c h o n d r i a ( S G D ) . T o further define the events g i v i n g r i s e to a - l i k e fakers i n the sovlA mutant, P C R w a s u s e d to detect the presence o f MATa  a n d MATa  l o c i i n the m a t e d products ( H u x l e y et a l . , 1990).  Interestingly, a l l m a t e d products f r o m the sovlA mutant c o n t a i n e d b o t h MATa l o c i , i n d i c a t i n g i n t r o d u c t i o n o f the MATa  allele into MAT'by  and  MATa  gene c o n v e r s i o n . T h i s w a s  not the general pattern o b s e r v e d i n w i l d - t y p e o r i n other mutants, w h e r e o n l y 3 % (1/39) or 6 % (24/386) o f isolates tested w e r e o f this type, r e s p e c t i v e l y . Interestingly, s o m e h i g h frequency a - l i k e fakers that s h o w e d w h o l e c h r o m o s o m e III l o s s f a i l e d to e x h i b i t a s e c t o r i n g p h e n o t y p e i n the C T F screen. O f 13 frequent A L F mutants a n a l y z e d i n F i g u r e 2.6, o n l y 5 w e r e i d e n t i f i e d b y C T F p h e n o t y p e i n the h i g h throughput screen: 3 w i t h strong (kar3A, siclA,  a n d dia2A) and 2 w i t h w e a k (rad27A a n d  nce4A) p h e n o t y p e s . T o c o n f i r m the presence o f assay difference, 5 frequent A L F mutants w e r e d i r e c t l y retested for the C T F p h e n o t y p e i n fresh transformants. T w o o f these (karZA, sicJA) e x h i b i t e d a strong C T F p h e n o t y p e as expected, and 3 s h o w e d m i l d s e c t o r i n g (esc2A, rad50A, xrs2A, F i g u r e 2.6c). T h u s , frequent A L F p r o d u c t i o n does not s t r i c t l y correlate w i t h frequent C F loss. T h i s c o u l d indicate that different factors i n f l u e n c e the inheritance o f endogenous c h r o m o s o m e III a n d the C F . O n e e x p l a n a t i o n i s that the telocentric structure o f the C F m a y enhance i n s t a b i l i t y i n s o m e mutants. A n o t h e r i s that the presence o f a p a r t i a l h o m o l o g o u s c h r o m o s o m e p r o v i d e d b y the C F m a y suppress i n s t a b i l i t y . F u r t h e r w o r k w i l l be r e q u i r e d to determine the u n d e r l y i n g b i o l o g i c a l m e c h a n i s m s that e x p l a i n these uncorrelated phenotypes.  2.3.5 Many yeast CIN genes are conserved C u r r e n t understanding o f m e c h a n i s m s that contribute to g e n o m e s t a b i l i t y has b e e n l a r g e l y fueled b y w o r k from m o d e l systems. T h i s a p p r o a c h has b e e n i n f o r m a t i v e for  60  human biology because o f the remarkable functional conservation within the chromosome cycle. To evaluate conservation o f yeast C I N genes identified i n the screens, B L A S T p searches using yeast amino acid sequences against proteomes from S. pombe, C. elegans, D. melanogaster, M. musculus, and H. sapiens were performed. A m o n g the 293 yeast C L N genes, 103 (35%) have homologues with e-values <10" i n all 5 organism 10  proteomes searched (see Appendix 5, which contains alignment results and functional summaries). Previous work showed that - 4 0 % o f yeast proteins are conserved through eukaryotic evolution (Rubin et al., 2000), and 30% o f known genes involved in human diseases have yeast homologues (Bassett et al., 1997). In agreement, 124 (42%) o f the yeast C I N genes identified i n this study have homologues i n human, with e-values <10" . 10  Human homologues o f yeast C L N genes represent candidates that may cause a C I N phenotype in human cells when mutated. Genetic perturbation causing a C L N phenotype can be a predisposing condition for cancer initiation or progression. A m o n g the 130 high confidence C I N gene list, 10 'top hit' human homologues (with e-values <10" ) (Table 10  2.3 and Appendix 6) have been previously shown to exhibit somatic mutations in cancer.  2.3.6 A strategy for cancer therapy: synthetic lethality and selective cancer cell killing While C L N mutations can contribute to tumorigenesis, the altered genotype o f a cancer cell may define a genetic "Achilles heel" that supports the selective killing o f tumor cells relative to adjacent normal cells. Genetic interactions resulting i n cell lethality hold promise for the design o f therapeutic approaches i n cancer. One kind o f genetic interaction with properties useful for this strategy is synthetic lethality, observed when two mutations individually capable o f supporting viability cause cell death when present together. Synthetic lethal mutant pairs identify genes that function i n parallel or related pathways that cannot be simultaneously lost (Ooi et al., 2006). Following this logic, cancer cells with a specific C I N mutation can be killed through loss o f function o f a synthetic lethal partner, while sparing normal cells (Hartwell et al., 1997; Kaelin, 2005). Systematic, large scale synthetic lethality analysis i n yeast provides a means for identifying such second-site loss-of-function mutations (Eason et al., 2004; Harris et al.,  61  2004; Pan et al., 2006; Tong et al., 2004). These budding yeast studies provide candidate human proteins whose inhibition (e.g., by a drug) may specifically k i l l tumor cells relative to normal cells. In this regard, gene deletions that exhibit synthetic lethality with multiple different C I N gene mutants are particularly attractive, as they might define broad-spectrum therapeutic targets. To address this concept, an analysis all known synthetic lethal interactions available for the yeast C I N genes that have cancer gene homologues (shown i n Table 2.3) was performed (8 o f the 10 have published synthetic lethal data). These 8 mutants are connected to 250 partners by 371 synthetic lethal interactions based on B i o G r i d (Stark et al., 2006) (data not shown). A m o n g the 250 partners, 61 bridge at least 2 yeast cancer homologues (Figure 2.7). Notably, 3 mutants (ctf4A, ctfl8A, dcclA) exhibit synthetic lethality with at least 6 cancer gene homologues. Interestingly, these 3 yeast genes share a role in sister chromatid cohesion (Mayer et al., 2004; Warren et al., 2004a). The 'hub' position o f these 3 mutants i n the interaction network implies that different C L N gene mutants share a common genetic vulnerability, and these common synthetic lethal interactors can serve as broad spectrum targets. The existant synthetic lethal dataset in budding yeast, although incomplete, is continuously expanding (Tong et al., 2004). Therefore, more 'hubs/common nodes' m a y b e identified. A comprehensive synthetic lethal network, together with an increased understanding o f the mutation spectrum in cancers, could provide insights pertinent to the design o f therapeutic approaches i n which human cancer cells are efficiently targeted for death by clinical intervention. Integration o f knowledge among emerging high throughput datasets in model organisms w i l l stimulate new research directions and applications in combating human diseases.  2.4 Discussion This work identified an extensive catalog o f genome instability mutants, based on phenotypic testing o f haploid and diploid yeast knockout collections for chromosome transmission fidelity ( C T F ) , bimater behavior ( B i M ) , and a-like faker formation ( A L F ) . This study characterized all non-essential yeast genes due to their accessibility for  62  phenotyping. Because many essential genes are known to contribute to genome stability from traditional approaches, a similar systematic screening effort for essential genes would be o f great interest, but w i l l first require the development o f a comprehensive hypomorphic mutation resource. A n extensive catalog is useful for the understanding o f mechanisms that maintain genome stability, for the identification o f new pathways important for genome maintenance, and for the organization o f functional networks. Systematic screening o f arrayed non-essential mutants avoids the sampling problem i n traditional mutagenesis methods, and supports the direct comparison o f phenotypes observed because all alleles are null. Differences i n both phenotype severity and assay specificity were observed. The relative contributions o f specific gene products to genome maintenance are revealed directly by phenotype strength. For example, rad27A, dial A, nce4A, and xrs2A exhibited the strongest A L F phenotypes (> 56-fold above w i l d type) among the high confidence yeast C I N genes, whereas well-studied damage response genes such as mec3A, mrclA, ddclA, and rad9A showed milder phenotypes (~11-fold). Apparently, under the growth conditions used in the screen, damage caused by the absence o f Rad27p, Dia2p, Nce4p, or Xrs2p proteins exceeds that resulting from checkpoint loss. In addition to phenotype comparisons within a given assay, results from different assays can be compared. For example, xrs2A exhibited one o f the highest A L F frequencies but a m i l d or absent C T F phenotype, indicating that the damage associated with xrs2A is more relevant to the maintenance o f a haploid chromosome III than to the artificial C F . In general terms, different chromosome marker stability assays ( C T F , A L F , and B i M ) defined both distinct and overlapping gene sets. The screen specificities could be due to sensitivity differences, but likely reflect mechanistic differences revealed by the assay systems. For instance, screening i n both haploids and diploids may give insight into how ploidy affects the maintenance o f chromosomes. The results demonstrate the importance o f using complementary assays to comprehensively identify genome maintenance determinants. The error observed i n the deletion arrays for non-essential genes underscore the importance o f mutant validation. It is widely known that mutant arrays are "evolving  63  resources" that accumulate changes due to manipulation and selective pressure (i.e. cross well contamination, aneuploidy, second site mutation, etc.). For example, 8% o f deletion mutants exhibit chromosome-wide expression biases indicative o f aneuploidy for whole chromosomes or chromosomal segments (Hughes et al., 2000). Lethal or slow-growth phenotypes caused by mutations in genes that do not segregate with a knockout allele occur at a frequency o f 6.5% (Giaever et al., 2002). However, parameters indicative o f array quality are usually not reported in studies using deletion sets. This issue becomes increasingly important as phenotypic data derived from distantly related replicates o f the deletion resource are compared and integrated. In this study, tag sequence analysis o f CLN mutant strains suggests false negative observations from well contamination were between 3 and 22% i n different screens (see Appendix 1 and 4 for details). This phenomenon is likely to be observed in other copies o f the deletion sets. Because the data were derived from 3 different array sets obtained from commercial distribution sources, and involved 2 laboratories both with experience i n handling large strain collections, it is unlikely that well address errors were due to laboratory specific manipulation o f the sets. A n empirical measure agrees: the C T F screen o f the knockout collection identified 12 o f 15 non-essential ctf mutants found previously i n a traditional mutagenesis (Spencer et al., 1990). T w o out o f the 3 missed mutants were due to incorrect strains at the well positions o f the array plates obtained from the commercial distribution source when checked by P C R . The false positive frequency due to secondary mutations or aneuploidy i n the deletion collection strains can also be estimated. For the haploid collections, the false positive rates were relatively high: l-{26X33%+(10+19)X91%+32}/86=18% for the MATa collection; l-{126X50%+(54+19)X91%+32}/231=31% for the MATa collection; and for the diploid collection, the false positive rate was lower: l-{26X75%+(10+54)X91%+32}/122=5%. These frequencies are consistent with the frequency o f unlinked recessive lethal mutations segregating independently o f the deletion mutations that was observed during construction o f the mutant resource (Giaever et al., 2002). In this study, false positive observations were rare among genes identified  64  in >1 chromosome marker loss assay (i.e. i n >1 deletion resource). The results were therefore partitioned into 130 high confidence genes (115 genes identified by >1 screen, plus 15 genes confirmed i n new transformants), and 163 lower confidence genes identified by single screens only. A full catalog o f yeast C I N genes w i l l provide a rich resource for ongoing studies of genomic instability in many organisms, including human. Additional screens o f nonessential yeast mutants (such as G C R screens in (Huang and Koshland, 2003; Smith et al., 2004)) and systematic incorporation o f essential mutants w i l l enhance the utility o f the yeast model system. Yeast C I N genes define cross-species candidate genes in humans that could contribute to C I N during tumorigenesis. A recent survey o f C I N colorectal tumors (Wang et al., 2004b) provides a proof o f principle. One hundred human candidate genes (chosen for similarity to model organism C I N genes) were screened for mutations in tumor samples, yielding 5 new C I N human genes mutated i n colorectal cancer (MRE11, ZwlO, Zwilch, Rod, and Ding, i n addition to the 2 previously known (CDC4, and BUB1) (reviewed in (Yuen et al., 2005)). These 7 C I N cancer genes account for <20% o f the C L N mutational spectrum i n colon cancer, and many other candidate C I N genes remain untested. Systematic analysis o f the mutational spectrum leading to a C I N phenotype in a model eukaryotic organism such as yeast w i l l therefore help to define the mutational spectrum leading to a C I N phenotype i n human cancer, and may accelerate the identification o f protein targets for selective killing o f cancer cells.  65  T a b l e 2.1 24 ctf mutants cloned to date A n additional 27 single member ctf isolates have not been cloned.  1 2 3 4 5 6  # alleles 30 11 11 8 5 5  7  5  CTF7/EC01  8  3  CTF 8  9 10  3 3  (not cloned) CTF10/CDC6  11  3  PDS5/SP027  12  3  CTF 12/SCC2/AMC3  X  13  1  CTF13/CBF3C  X  14  1  CTF14/NDC10  X  15  1  CTF15/RPB4/SEX3  16 17  1 2  (not cloned) CTF 17/CHL4/MCM1  18  3  CTF18/CHL12  19  2  CTF 19/MCM18/LPB  ctf  s3 si 27 sl38 S141  Gene name  Essential?  CTF1/CHL1/LPA9 (not cloned) CTF3/CHL3 CTF4/POB1 /CH L15 CTF5/MCM21 CTF6/RAD61  Cohesion  X  X  BIM 1 /HSN9/YEB1 1  SIC1  1 1  SPT4  S143  Function  NUP170/NLE3 MAD1  sl55 si 65  1 1  MCM16 SCC3/IRR1  X  sl66  1  SMC1/CHL10  X  27  109  Total  7  Central kinetochore Cohesion Central kinetochore Cohesion Cohesion (establishment) Cohesion (alternative RFC) DNA replication Cohesion/condensati on Chromosome condensation Inner kinetochore (CBF3) Inner kinetochore (CBF3) Subunit of RNA polymerase II Central kinetochore Cohesion (alternative RFC) Central kinetochore Microtubule-binding at SPB/kinetochore Cell cycle regulator Chromatin structure/ transcription Nucleoporin Kinetochore protein /spindle checkpoint Central kinetochore Cohesion Cohesion/condensati on  Cohesion  DNA/RNA metabolism  X X X X X X X X X X X X X X X X X X X X X X X X 10  66  Kinetochore  9  5  T a b l e 2 . 2 L i s t o f yeast strains u s e d i n C h a p t e r 2 The genotypes and origins of strains used in this study are shown. Strain Genotype BY4741 MATa BY4742 MATa Y2454 MATa mfalA::MFAlpr-HIS3 canlA ura3A0 his3Al  YPH1724  lys2A0  MATa  ade2-101::natMX  ura3A0  leu2A0  YPH255  MATa  YPH1124  MATa  YPH1725  MATa  Ieu2-Al Ieu2-Al  ade2-Wl  his3-A200  ura3-52  his3-A200 natMX  MATa  ade2-101::  mfalA::MFAlpr-HIS3  YPH315 YPH316 YPH1738  MATa  hisl  MATa  hisl  MATa/MATa his3Al  lys2-801  trpl-A63  Hieter lab  SUP11 ura3-52  CFIII(CEN3.L)::URA3 ade2-101::  This study  lys2A0  CFVII(RAD2.d)::URA3 ade2-101  canJA  mfalA::MFAlpr-HIS3  his3Al  lys2-801  trpJ-A63  (Pot et al., 2003)  SUPU  his3  mfalAr.MFAlpr-HIS3  YPH1726  leu2A0  Reference (Brachmann et al., 1998) (Brachmann et al., 1998) (Tong et al., 2001b)  ura3 lys2  canlA  This study  CFVII(RAD2.d)::URA3.SUP11 natMX  his3  ura3  lys2  CFIII(CEN3.L)::URA3  ura3A0/ura3A0  LYS2/lys2A::kanMX6  leu2A0/leu2A0  can]A  his3Al/  MET15/metl5A::kanMX6  67  This study  SUP11  (Spencer et al., 1990) (Spencer et al., 1990) This study  T a b l e 2.3 H u m a n p r o t e i n s h o m o l o g o u s to yeast C I N genes are m u t a t e d i n c a n c e r The protein sequences corresponding to 130 high confidence yeast CIN genes were used as queries in a BLASTP search against the human RefSeq protein database. Online Mendelian Inheritance in Man (OMIM, www.ncbi.nlm.nih.gov/omim) and cancer census (Futreal et al., 2004) databases were used to identify cancer associated mutations in 'top hit' human genes.  Yeast Gene  Top Human Hit  ADE17  ATIC  RAD54  RAD54L  1E-164  RAD51  RAD51  1E-122  RDH54  RAD54B  1E-121  SGS1  BLM  1E-115  MRE11  MRE11A  1E-108  DUN1  CHEK2  6E-55  BUB1  BUB1  1E-41  MAD1  MAD1  5E-12  CDC73  HRPT2  9E-12  E-value 0  Disease Description, MIM#(disease) Anaplastic large cell lymphoma  MIM# (gene)  Non-Hodgkin lymphoma; Breast cancer, invasive intraductal; Colon adenocarcinoma susceptibility to Breast cancer, 114480 Non-Hodgkin lymphoma; Colon adenocarcinoma Bloom syndrome, 210900  603615  Cancer census OMIM  179617  OMIM  604289  OMIM  604610  Ataxia-telangiectasia-like disorder, 604391; Colorectal cancer  600814  Li-Fraumeni syndrome, 151623; Osteosarcoma, somatic, 259500; susceptibility to Breast cancer, 114480; Prostate cancer, familial, 176807; susceptibility to colorectal cancer Colorectal cancer with chromosomal instability Lymphoma, somatic; Prostate cancer, somatic, 176807 Hyperparathyroidism-jaw tumor syndrome, 145001; Hyperparathyroidism, familial primary, 145000; Parathyroid adenoma with cystic changes, 145001  604373  OMIM, Cancer census OMIM, (Wang et al., 2004b) OMIM  602452  OMIM  602686  OMIM  607393  OMIM  68  Reference  F i g u r e 2.1 T h r e e screen m e t h o d s a) C T F screen m e t h o d  The genotypes of the donor strain and the MA Ta deletion mutants are shown within the schematic yeast cell respectively. The gray line with a gray circle depicts the CF. Each filled circle represents a selectable marker used in the SGA scheme, whereas an open circle in the corresponding color represents the unmarked allele. The ' X ' indicates mating between the 2 strains. The arrows indicates the selection procedures. Yeast cells enclosed by the dashed rectangles represent cells that were selected. The dashed arrow indicates the loss of a CF. Examples of severe and mild sectoring colonies, as well as wild-type colonies are shown. b ) B i M screen m e t h o d  An example of an agar plate containing homozygous deletion mutants grown in 96-array format is shown. The yellow rectangles represents agar plates containing the specified media, and the indicated yeast strains were grown on them. The arrows indicates replica plating, pointingfromthe source plates to fresh destination plates. The blue blocked arrows indicate scanning, visual inspection and densitormetry reading of the Sc-6 plates, which selected for mated products. An example of a set of plates mated with MATa and MATa mating testers, respectively, is shown. Yeast cells enclosed by yellow squares had high mating with both MATa and MATa mating testers; whereas yeast cells enclosed by red and green squares had high mating with MATa and MATa mating testers, respectively, and these were not included in further analysis. The bottom shows an example of a retested plate containing 3 homozygous deletion mutants, each represented by 4 independent colonies, together with the wild-type diploid (YPH1738) and chllA/chllA. On the left is the SC-6 plate replica plated from the YPD plate with MA Ta mating tester (X MATa), and on the right is the SC-6 plate replica platedfromthe YPD plate with MA Ta mating tester (X MA Ta). c) A L F screen m e t h o d  The scheme of a-like faker selection is shown in the box, with the genotypes of the starting and selected strains indicated. The actual patching and replica plating procedures are shown below. An example of SC-6 plate with mated products derivedfrommating of the MA Ta mating tester with 12 deletion mutants, positive and negative controls on the same plate, is shown. bub3A, hst3A and yor024wA showed elevated matingfrequencieswith the MATa mating tester. The retest plate of these 3 mutants, each represented by 4 independent patches, is shown.  69  Figure  2.1a CTF screen method  Donor strain (YPH1725 or YPH1726)  MA Ta deletion mutants  Mating on Y P D (1 day) (25°C for all incubation)  X Diploid selection by SC-URA+G413 (twice: 2 days & 1day)  Sporulation (9 days) Examples of spore genotypes  Select MA Ta haploids with CF using Sc-HIS-UFtA-ARG+can (2 days)  Select MATa haploids with CF, ade2-101 &ykoA by Sc-HIS-URA-ARG+can+G418+clonNAT (twice: 2 days each)  Streak on media non-selective for CF (Sc with 20% A D E ) t o examine sectoring phenotype (6-7 days at 25°C &5-7 days at4°C)  Loss of CF  Severe sectoring  Mild sectoring  70  Wild-type  F i g u r e 2.1b B i M screen method  homozygous diploid deletion mutants in 96-arrayfonrat on YPD+G418 (3 days)  Replica plating  imTa vkoAhis3 msi Mf\Tay*oAhis3 HSS1  YPD+G418  Example of a 96-array on SC-6 media selectim for mated products  Sc 6 (background control) MA Ji HIS3 hisl  mat rig tester KYPH315) lawn MAT*H)S3 Ms1  mat rig tester tYPH 316) lawri.  Ljjvlating on YPD (2 days)  floating on YPD (2 days)  Mated productj selection on Sc-6 (3 days) Scanning, visual inspection Mated product] & densitometry reading selection on Sc-6 High mathg rate (3 days) with MATa only  1  •  High mating rate wM  •  MATa AND MATa  High mathg rate with MATaonfy  Retest of candidates using 4 independent colonies: example  X MATa  X MATa  71  i i—i  I—i .  rare  F i g u r e 2 . 1 c A L F screen method  MATaykoA his3 HIS1  Q"jD  Loss of MATa locus |  [null] ykoA his3 HIS1 (a-likefaker cell) \^J^  C JO MATa HIS3 hi si (mating tester)  1 (  JD  MATa YKO HIS3 hisi (His+ mated product)  MATaykoA  his3HIS1  P a t c h e s ( Y P D overnight)  R e p l i c a o n t o l a w n of MATahisI  mating tester  ( Y P D overnight)  Fold Change  Calculation  m u t a n t m e d i a n \4 p a t c h e s l wt m e d i a n [all w t p a t c h e s ] Re-phenotype candidates using 4 independent colonies  I  72  Figure 2.2 Three marker loss screens A. Haploid yeast knockout mutants (ykoA) containing ade2-101 and a chromosome fragment (CF, blue line whose centromere is depicted as a circle) were generated. The CF contains the SUP I1 gene (blue rectangle) on the telocentric arm. ade2-l0l cells develop red color, but the SUP 11 gene on the CF suppress the pigment formation (Gerring et al., 1990; Hegemann et al., 1988; Warren et al., 2002). Cells that contain the CF are therefore unpigmented, whereas cells without the CF are red. Mutants that inherit the CF unstabliy form colonies with red sectors. An example was shown. The chromosomal context of the tested strains was shown at the bottom. Loss of SUP11 is caused by loss of the whole CF. B. Homozygous diploid yeast knockout mutants were tested for 'bimater' phenotype. For example, loss of the MA Ta allele (depicted in gray) causes the development of an a-type mating cell, which is detected by its ability to mate (depicted by 'X') with a MA Ta tester strain containing complementing auxotrophy to support selection of mated diploids. Mutant strains exhibiting unstable inheritance of the MA T locus will lose either allele in individual cell and exhibit a 'bimater' phenotype in a population. The mutants in the squares show elevated formation of mated cells after exposure to either MA Ta or MA Ta testers (Spencer et al., 1990). The heterozygous MAT locus in the homozygous diploid knockout strains was shown. Loss of heterozygosity of the MAT locus can be caused by various mechanisms indicated C. MA Ta haploid yeast knockout mutants were tested for elevated frequency of a-like faker cells. Loss of the MA Ta locus (depicted in gray) in haploids results in dedifferentiation to a-mating type. The presence of these cells is detected by selection for mated products after exposure to a MA Ta tester strain. Examples of mated products formed from a wild-type and an alf mutant were shown  CF Chrlll  5 UP11  MATa  Chr III  MATa  Chr I  MATa  MATa  C h r o m o s o m e transmission Diploid Bimater ( B i M ) fidelity ( C T F ) Chromosome Loss Chromosome Loss Rearrangement Gene Conversion Mitotic Recombination  73  A-Like Faker (ALF) Chromosome Loss Rearrangement Gene Conversion  F i g u r e 2.3 130 high confidence non-essential yeast C I N genes The Venn diagram shows the distribution of 293 mutants identified initially across the 3 screens. The numbers in parenthesis denote single-assay knockouts confirmed in independent transformants, which are included in the detailed diagram to the right. The detailed diagram to the right summarizes the 130 high confidence genes described in Appendix 1 (For the other 163 genes, see Appendix 2). All gene names are connected to 1 or more of the 3 screen nodes (CTF, BiM, or ALF), indicating the screen phenotypes the knockout mutants exhibit. Gene names in black typeface are those fully validated in all 3 deletion arrays: i.e. tag sequencing indicated the presence of only the correct mutation. For these mutant, a present or absent CIN phenotype observed in any screen is meaningful. Gene names in blue italic typeface failed tag validation in at least 1 of the deletion collections, and therefore phenotype information is missing from at least 1 screen. These partially characterized genes are placed to indicate positive phenotypes known from validated deletion sets (Appendix 1 and Appendix 2 contain details of the validation status). The node colors indicate biological process. Genes associated with more than one functional group is represented by the one highest in the color key for simplicity.  CNN1^  PS64  J'IG1  ji^oyyj  126(11)  K  A  R  3  GR064W' CTF19  CIN8  L010W-A • U010C-©'  YKL0S3'  'LUOC PL017C  ALF  IUP133  (122 total)  (231 total)  Biological Process Chromosome segregation 41 Cell cycle DNA repair, DNA damage • response, DNA recombination 41 DNA replication sjjk DNA metabolism, ™ chromosome organization 4) Transcription 41 Transport  3  JBR113W  41 Protein metabolism 4> Metabolism  MRE11  TAT  ®£* WWSS1  4) Oxidative stress response  •ADE6  41 Unknown  L32 P A 1 2  74  ESC2  1^RM3  (  ^  I  F  T  1  F i g u r e 2.4 Functional groups o f 293 C L N genes GO biological process terms for the 293 marker loss genes were obtained using Osprey (vl.2.0) functional groupings (Breitkreutz et al., 2003b). For genes associated with more than one GO biological process, a single GO process was assigned according to the priority shown in the Figure 2.3 color key for simplicity.  75  F i g u r e 2.5 92 protein interactions among 102 CIN proteins  Different types of protein interactions were shown among CIN proteins using GRID database and OSPREY program, indicated by arrows pointing from a bait/query. Color of the proteins represents the GO biological process. Known complexes or pathways were circled and annotated in red. Abbreviations: chkpt. (checkpoint); rec. (recombination); C t r l , (control); mod. (modification).  Q Con Or oanutatwn and Braqenesls CeS Growth andor mamtenanca Prefoldin complex 0  (tubulin folding)  Microtubule  &  / _ _ \  5iLipi2<a<yp84j*jpi33 "jpore  N U P 1 2 0 N U P 8 4 NI  AlternativeSl« RFC (cohesion) Mitoj  y  Protein  sphotase^ complex  ISM6 . LSM1 nrj RNA splicing  ft SPT2  BRL3  IEA1 KGD1  JGR058W JTNL047C GRXS YDR279W YLR154C JCROSSg YPUH7W Transcription Ubiquitination  DNA  Post-replication repair  ADE2 . SEO ^ ^ 3 , • ^ — * 4  /""jJ6R1 /  , damage chkpt  W ^ i  RADlS\  DSB repair RTT101  MMS22 ~ ESC4  YLR320t^  MFT1  5  at  /RAMI  RA06S  £ Protein Degradation Transcription II DKA Replication Protein toosytinnests  complex  igsomal  —  cycle ctrl.  DNA damage*  mmmm  Nuclear  f  YOR066^^IC1 Cell  «wn»a)«»i #  s  " RRM3  Dtunap»  0 ProteeaanrtnoecMldiowhoiyWion DHADamaoeRewonM 0 prMeet transport 0 Transport % RNALocabauon Sponuuin s§l nuprricetiina 0 R*OMrn« Bionimesis 0 Carbonydrate uatabMism  THPiTHI'  (3 CI con iplex _ YyI » c B R i n L n n l P x  e r l f n e n t a l  E  MUS81  .2  RAD59  «riM1^Vr5R100wV ' "Rec repair'  ,§c. repair  ~3P  CDC73  ASF1  ITIOd.  •  AT fBTITY CHROMATOGRAPHY PUNFIED COMPLEX I M M M  76  Sys^ms  MWenYPreCKTMION  F i g u r e 2.6 A - l i k e fakers result from whole chromosome loss, gross chromosomal rearrangement, and gene conversion A. Individual colonies selected after mating were characterized using pulsed-field gel electrophoresis (top, ethidium bromide stained gel) and in-gel hybridization with a radiolabeled probe (bottom, autoradiogram) that hybridizes chromosomal bands containing the mating type locus and/or silent mating type loci located distally on each arm. Chromosomes III from the mating tester and deletion mutant were of distinct size (top and bottom chr III bands, respectively). In some strains, a less intense signal for rearrangement chromosomes reflects poor mitotic transmission or hybridization only to HMRa which has imperfect homology to the radiolabeled probe. B. Summary of electrophoretic karyotypes from 13 ALF mutants. ALF frequency is shown as fold over wild type. Event percentages (chr III loss, gross chromosome rearrangement (GCR), or retention of normal structure) are calculated from independent wild-type or mutant mated products (n=40 and n>10, respectively). The outcome distributions for sovlA and rad27A are significantly different from wild type (chi square, p < 0.01). C. Discordant CTF sectoring phenotypes are observed in knockout mutations with similar ALF frequencies.  Ill m  *  Strain Wild type rad27A dia2A ybrll3wA nce4A xrs2A esc2A top3A kar3A rad50A siclA adelA omalA sovlA  m  Fraction Fraction Fraction ykoA ykoA ykoA ALF Fold Chrlll Chrlll Chrlll Change Loss GCR Retained 1 0.68 0.20 0.12 63 0.30 0.60 0.10 60 0.70 0.30 0.00 56 1.00 0.00 0.00 56 0.70 0.20 0.10 48 0.14 0.86 0.00 43 0.50 0.30 0.20 42 0.60 0.40 0.00 40 1.00 0.00 0.00 0.64 36 0.29 0.07 31 0.70 0.30 0.00 31 0.70 0.20 0.10 30 0.80 0.10 0.10 8 0.00 0.00 1.00  77  F i g u r e 2 . 7 . C o m m o n synthetic lethal interactions among yeast C I N genes that have human homologues mutated in cancer Eight yeast CIN genes with top hit human homologues mutated in cancer (e-value <10" ) are also found in the public interaction database BioGrid (Stark et al., 2006). They are placed peripherally, and shown in black. 61 interactors that have at least 2 synthetic lethal connections with the yeast cancer homologues are shown. The arrows point from a query to a target gene hit in the synthetic lethal screens. The 27 genes in blue fonts are themselves high confidence CIN genes identified by the screens, and 12 genes in purple fonts are in the lower confidence CIN gene list. The 3 genes that have 6 common synthetic lethal interactions 10  78  CHAPTER 3 Identification of Somatic Mutations in Cohesion Genes in Colorectal Cancers with Chromosome Instability  A modified version o f this chapter has been prepared for publication. Karen W . Y . Yuen*, Tom Barber*, Marcelo Reis*, K i r k M c M a n u s , Forrest Spencer, Bert Vogelstein, Victor Velculescu, P h i l Hieter, and Christoph Lengauer (*These authors contributed equally to this work). Identification o f Somatic Mutations in Cohesion Genes i n Colorectal Cancers with Chromosome Instability.  79  3.1 I n t r o d u c t i o n  While the majority o f colorectal cancers exhibit C I N , the molecular and genetic basis for this phenotype is not well characterized. Over the last decade, only a handful o f genes known to be important for maintaining chromosome stability ( C I N genes) have been systemically tested and identified to have mutations i n colorectal cancers, including BUB1, BUB1B (Cahill et al., 1998), CDC4 (Rajagopalan and Lengauer, 2004b), MRE11A,  ZwlO, Zwilch, Rod and Ding (Wang et al., 2004b). A l l these genes were first  identified based on chromosome segregation or cell cycle phenotypes i n model organisms such as yeast and fly. These cross-species connections are examples o f the high degree o f evolutionary conservation in basic cellular mechanisms, and how basic biology studies in model organisms can be applied efficiently to gain an understanding o f human disease. Recently, germline biallelic mutations in BUB IB have been associated with mosaic variegated aneuploidy and inherited predispositions to cancer, strongly supporting a causal link between C I N and cancer development (Hanks et al., 2004). However, each o f the 8 genes mentioned above accounted for only a small fraction o f the somatic mutation spectrum in colon cancer (1-10%), suggesting that more C I N genes could each be mutated to cause C L N in cancer. Systematic mutational analysis o f C I N genes i n colorectal cancers would therefore be useful to determine the complete mutational spectrum and mutation frequency leading to C I N . Indeed, the Lengauer/Vogelstein groups sequenced 100 candidate C I N genes (based on their similarity to yeast and fly genes) and identified 5 o f the 8 genes mentioned above, suggesting that expansion o f this kind o f study would lead to the identification o f additional relevant C I N genes (Wang et a l , 2004b). A m o n g the 100 candidate human C I N genes pursued i n the study, the best yeast homologue o f 30 human CLN genes yields the same human genes b y reciprocally searching the best human homologue b y B L A S T p (Table 3.1), outlining the number o f yeast genes that have been used previously to identify human C I N gene mutation i n cancer. In this study, the potential role o f C I N genes i n a panel o f colorectal cancers was systematically analyzed by sequencing 101 candidate human C I N genes based on their  80  similarity to yeast homologues. 20 somatic mutations were identified genes that function in 4 functional groups. Seventeen o f the mutations were found i n 5 genes that are directly involved i n sister chromatid cohesion (SMC1L1,  CSPG6, NIPBL,  STAG2, and STAG3).  Furthermore, single somatic mutations were identified in each o f these 3 genes: BLM, the B l o o m syndrome gene; RPN20, a E3 ubiquitin ligase; and UTX, a transcription factor. This study broadens the mutational spectrum o f C I N genes i n colorectal cancer. The results are consistent with a genetic basis for C L N , and with C L N having a role i n tumorigenesis. Phenotypic analysis o f a conserved missense mutation i n yeast SMC1 revealed a modest recessive C L N phenotype i n yeast cells. Further functional studies o f the somatic mutations found w i l l enhance our understanding on whether these mutations cause C L N i n cancer cells.  81  3.2 Materials and Methods 3.2.1 Gene identification 293 non-essential yeast C I N genes identified from recent comprehensive genomewide screens (Yuen et al., submitted; see Chapter 2) were searched for human homologues by B L A S T p using Refseq database, and 88 human genes were selected based on the extent o f sequence similarity (63 had a e-value <1E-10) and strength o f the CLN phenotype in yeast. 64 were derived from the high confidence list (including 2 homologues o f CHL1) and 24 were from the low confidence list. 2 non-essential CTF genes identified by traditional random mutagenesis (Spencer et al., 1990) but missed i n the high-throughput screens (NUP170, RPB4) were also included; as w e l l as 11 human genes homologous to essential yeast genes involved i n chromosome transmission fidelity (Spencer et al., 1990) and cohesion. A total o f 101 candidate C I N genes were analyzed.  3.2.2 Sequencing P C R primer design, amplification, sequencing, and sequence analysis were performed as previously described i n (Sjoblom et al., 2006; Wang et al., 2004b).  3.2.3 Yeast smcl mutants construction and characterization Mutations were introduced into the yeast SMC1 gene by performing two rounds of P C R . One set o f primary P C R product was amplified from wild-type (WT) genomic D N A using a sense primer ~500b upstream o f the mutation, and an antisense primer containing the mutation i n the middle; and a second primary P C R product was amplified with a sense primer containing the mutation i n the middle and an antisense primer ~200b downstream o f SMC7 that included a 20b homology to the T E F promoter. The primary P C R products were gel purified and used as template for a secondary round o f P C R using the sense primer ~500b upstream o f the mutation and the antisense primer ~200b downstream o f SMC7 that included the 20b homology to the T E F promoter. The secondary P C R product was gel purified, cloned i n Topo2.1 vector and sequenced. The insert was cut with flanking restriction enzymes and co-transformed with a P C R product  82  containing k a n M X , with flanking T E F promoter and terminator sequences, into a WT diploid strain (YPH986) containing C F I I I ( C E N 3 . L ) . Transformants were selected on G418 and checked by colony P C R . The mutation was sequenced i n the heterozygotes by P C R amplification with a primer upstream o f the mutation and a kanMX-specific antisense primer. The heterozygotes were sporulated, and dissected to isolate haploid spore clones. The chromosome transmission fidelity (ctf) phenotype o f both the heterozygous diploids and the haploids were checked at 25°C, 30°C, and 37°C (Spencer et al., 1990). Quantitative ctf assays were performed by counting the frequency o f halfsectored colonies i n haploids at 37°C on S C media with 20% adenine concentration as in (Shero et al., 1991). Yeast strains used i n this study are listed i n Table 3.2. A t least 3000 cells were plated, and the experiment was done i n duplicate.  83  3.3 Results 3.3.1 20 somatic mutations were found in 8 CIN genes Based on recent comprehensive genome-wide screens o f the yeast non-essential gene deletion set for C L N mutants (Yuen et al.; see Chapter 2), and previously identified essential yeast C L N genes identified by traditional random mutagenesis (Spencer et al., 1990), a list o f yeast C L N genes was complied and used to identify their human homologues based on protein sequence similarity. The list was prioritized based on the extent o f yeast/human similarity and phenotype strength in yeast, and 101 human candidate C I N genes were selected for somatic mutation detection i n a panel o f 36 colorectal cancers (Table 3.3). In total, 1066 exons encode these 101 candidate genes, and corresponding primer pairs were used to P C R amplify these exons. After excluding known polymorphisms present i n the human genomic database, all novel variants were resequenced using matched normal D N A from the patient to distinguish true somatic mutations from pre-existing variants. Somatic mutations in 8 genes (SMC7LI, NIPBL,  CSPG6,  STAG2, STAG3, BLM, UTX, and RNF20) were identified, 5 o f which are  involved in sister chromatid cohesion. To assess the frequency at which the genes are mutated in colon cancer, mutational analysis was expanded to an additional 96 colorectal cancer samples for 4 o f the 8 genes (Table 3.4). This revealed that three o f the cohesion genes, SMC 1 LI, CSPG6 and NIPBL,  have a mutation frequency o f - 3 . 8 % i n colon  cancers.  3.3.2 Mutation frequency in comparison to prevalence of mutations The identified somatic mutations could represent either 'passenger' mutations that occur as a consequence o f tumorigenesis, or 'functional' mutations that underlie tumorigenesis. Mutations i n genes with functional relevance are expected to occur at a frequency higher than random chance. Assuming that there is ~1.5kb o f coding sequence per gene, ~5.4Mb was sequenced i n the initial screening o f 101 genes i n - 3 6 cancers (1.5kb X 101 genes X 36 tumors). A study by W a n g et al. indicated that - 1 nonsynonymous somatic change accumulates per M b o f C L N tumor D N A , suggesting that  84  the mutation rate i n C I N tumor cells is similar to that i n normal cells and that most sporadic colorectal cancers do not display a mutator phenotype at the nucleotide level (Wang et al., 2002a). Another study found 151 mutations i n 250Mb o f D N A for a mutation rate o f 0.6 changes per M b (Bert Vogelstein, unpublished). These data have significant implications for the interpretation o f somatic mutations i n candidate tumorsuppressor genes. Based on this estimation, ~3-5 mutations are expected from the first pass sequencing. Indeed, 8 somatic mutations were identified, suggesting that at least some (or all) o f the mutations identified could be passenger mutations. In order to accurately determine the mutation frequency, the sequencing o f 4 genes identified i n the initial round was scaled up to an additional 96 tumors. A m o n g the 4 genes, SMC 1 LI, CSPG6, and NIPBL are involved i n cohesion, and one (BLM) functions in D N A repair. For each o f these 4 genes, ~200kb genomic D N A was sequenced (1.5kb X 132 tumors). Based on the 1 mutation/Mb estimation, 0.2 mutations are anticipated for each gene. 5 mutations were identified i n each o f the 3 cohesion genes, which is 19 times higher than expected. Such non-randomness i n mutation pattern suggests that the mutations i n cohesion genes are o f functional relevance rather than "passenger" changes. However, no additional mutation was identified for BLM.  3 . 3 . 3 A conserved missense mutation in yeast SMC1 causes mild CIN In order to elucidate whether the mutations found can lead to C I N , human SMC1L1 was aligned with yeast SMC1 (Fig. 3.1a), and the analogous mutations found i n human SMC 1 LI were constructed i n yeast SMC1 (Fig. 3.1a,b). The smcl mutants were then assayed for chromosome transmission fidelity (CTF) b y monitoring the loss o f an artificial chromosome fragment in heterozygous diploid and haploid strains. The 1877 nonsense mutation, which results i n truncation o f the C-terminal region, led to lethality in a haploid background. VI1871, one o f the 3 missense mutations is i n the conserved ATPase domain at the C-terminal region. In haploids, this conserved mutation caused a 2-fold increase i n chromosome loss at 37°C (Fig. 3.1c). Antisense inhibition o f SMC1L1 in human fibroblast cells has been shown to lead to aneuploidy and chromosome  85  aberrations, as well as an increased frequency o f micronuclei formation and apoptotic cells i n long-term cultures (Musio et al., 2003).  86  3 . 4 Discussion  3 . 4 . 1 SMC1L1,  CSPG6, NIPBL,  STAG2 a n d STAG3  SMC 1 LI, CSPG6 and NIPBL (Delangin/SCC2)  were each found to be mutated in  5 o f the 132 tumor samples analyzed (i.e. a frequency o f - 3 . 8 % for these genes), and STAG2 and STAG3 each had a single mutation i n 36 tumors sequenced. SMC1L1 and CSPG6 (SMC3), together with SCC1 (MCD1IRAD21)  and SCC3 (STAG1, STAG2 and  STAG3 isoforms i n vertebrates), form the essential cohesin complex, which is required for cohesion o f sister chromatids and for accurate chromosome segregation (Tanaka et al., 2000). NIPBL,  together with SCC4, forms an essential complex that loads cohesin to  replicated sister chromatids during D N A replication (Ciosk et al., 2000). Recently, NIPBL and SMC1L1 were found to be mutated in Cornelia de Lange ( C d L ) syndrome, characterized by facial dysmorphisms, upper limb abnormalities, growth delay and cognitive retardation (Krantz et al., 2004; M u s i o et al., 2006; Tonkin et a l , 2004). NIPBL is expressed ubiquitously, but with variable tissue-specific expression. NIPBL not only has a role i n cohesion, but also functions in developmental regulation b y affecting gene expression (Rollins et al., 2004); whether these functions are independent from each other, and how much NIPBL expression is required for each function remains unknown. Recently, CTF7IESC02,  an acetyltransferase required for cohesion establishment i n S  phase, was found to be mutated i n Roberts syndrome and S C phocomelia, which has several phenotypes overlapping with Roberts syndrome (Schule et al., 2005; V e g a et al., 2005). Precocious sister chromatid separation has been described i n C d L syndrome, Roberts syndrome, and mosaic variegated aneuploidy, and various cancers (Kaur et al., 2005). However, patients o f C d L and Roberts syndromes do not have cancer predisposition. The present study is the first report identifying mutations i n genes functioning in cohesion in human cancers.  3 . 4 . 2 BLM BLM was found to be mutated in one out o f 132 tumor samples analyzed. BLM, together with WRN and RECQL4,  are homologous to yeast SGS1 i n the R e c Q helicase  87  family, and all 3 genes are mutated in cancer prone syndromes (see Table 1.1). BLM is a D N A structure-specific helicase, which plays a role i n the resolution o f D N A structures that arise during the process o f homologous recombination repair, by catalyzing Holliday-j unction branch migration and annealing o f complementary single-stranded D N A molecules (reviewed i n (Cheok et al., 2005)). In the absence o f BLM, cells show genomic instability and a high incidence o f sister-chromatid exchanges. Gruber et al. (Gruber et al., 2002) determined that carriers o f the BLMiAsh) founder mutation (causing frameshift and truncation) have an increased risk o f colorectal cancer, and they also observed a low frequency o f the BLM(Ash) mutation in lymphoma, breast, ovarian, and uterine cancers. M i c e heterozygous for a null mutation of BLM develop lymphoma earlier than wild-type littermates in response to challenge with murine leukemia virus, suggesting that BLM haploinsufficiency is associated with tumor predisposition (Goss et al., 2002). However, further functional analysis is required to determine whether the heterozygous missense mutation found i n this study causes a C L N phenotype.  3 . 4 . 3 RNF20 One heterozygous missense mutation was found i n RNF20, which encodes a E3 ubiquitin ligase. RNF20 forms a complex with RNF40, interacts with an ubiquitin E 2 conjugating enzyme UBCH6, and establishes H2B lysine 120 monoubiquitylation, which is associated with transcriptional activity (Pavri et al., 2006; Z h u et al., 2005). This modification subsequently regulates H2B methylation and expression o f homeobox ( H O X ) genes, which are required for proper development. In yeast, H2B ubiquitylation by RAD6(E2)-BRE1(E3)  has been shown to be required for the D N A damage checkpoint  response (Giannattasio et al., 2005). Interestingly, yeast-two-hybrid analysis o f the yeast homologue o f RNF20, BRE1, indicated that it interacts with the coiled-coil region o f 3 proteins in the structural maintenance o f chromosomes family (SMC1, SMC2, SMC3) and some kinetochore components (SLK19, NUF2) (Newman et al., 2000), and may play a direct role in chromosome maintenance.  88  3.4.4  UTX One heterozygous missense mutation was found i n UTX. UTX is a transcription  factor that contains the tetratricopeptide repeat (TPR) motif. The yeast homologue SSN6ICYC8, together with TUP I, is involved i n histone deacetylation, which is associated with transcriptional repression. SSN6 functions as a negative regulator o f the expression o f a broad spectrum o f genes (reviewed in (Smith and Johnson, 2000)), which explains why the phenotype of ssn6 mutant is pleiotropic. ssn6 mutants exhibits a modest effect on the maintenance o f minichromosomes (Schultz et al., 1990).  While it is evident that the role o f cohesion genes and BLM i n chromosome maintenance is w e l l conserved, it is still unclear whether RNP20 and LVTXplay a direct role in chromosome maintenance i n yeast or i n human. Further functional studies in model organisms and in mammalian cells w i l l delineate the roles o f these candidate C I N genes, reveal whether the mutations cause C I N , and elucidate the degree o f functional conservation. This study initiated the characterization o f the cancer somatic mutations i n SMC 1 LI by introducing the corresponding mutations i n yeast SMCL  The conserved  mutation caused a mild C I N phenotype in haploids. Although the heterozygous diploids of this conserved mutation or the truncation do not exhibit a detectable C I N phenotype in yeast, it is possible that human cells may be more sensitive to perturbations i n C L N genes, due to larger chromosome and genome size. A precedent for this hypothesis is that mitotic checkpoint components are dispensable in yeast, but are essential i n higher eukaryotes (Babu et al., 2003; Baker et al., 2004; D a i et al., 2004; Kitagawa and Rose, 1999; Kops et al., 2004; M i c h e l et al., 2001). The results presented here broaden the mutational spectrum o f colorectal cancers, and are consistent with previous observations that each C I N gene is mutated at a low frequency (-3.8% for each o f the 3 cohesin related genes). Therefore, a variety o f C I N genes could each be responsible for a small proportion o f cancers. C L N is a hallmark o f most solid tumors, so it w i l l be o f interest to compare the mutational spectrum for  89  colorectal cancers to that o f other types o f solid cancers. The technical information gained in colorectal cancer w i l l be useful for similar analysis i n other tumor types. Mutational spectra may also allow classification o f tumors, which could have implications for improved diagnosis, prognosis, or predictions o f response to therapy. For example, the Vogelstein group recently pursued another large-scale mutational analysis o f 13,023 genes in 11 breast and 11 colorectal cancers (Sjoblom et al., 2006). This unbiased study provided an estimate o f the total number o f nonsynonymous mutations that arise in a typical cancer, thereby allowing statistical differentiation between passenger mutations and mutations with functional implications. Their study revealed 189 genes that were mutated at significant frequency. These cancer genes encode a wide range o f cellular functions, including genes that have been shown to be somatically mutated or implicated i n tumorigenesis i n expression studies, but also many genes that were not previously suspected to contribute to the pathogenesis o f cancer. Interestingly, that study also identified substantial differences i n the mutational spectra in different tumor types. K n o w i n g the mutational spectra i n cancers would be useful for therapeutic design. A s described in Chapters I and II, a second-site loss-of-function mutation that causes synthetic lethality with a C L N mutation can lead to selective killing o f tumor cells. Combining synthetic lethal data available i n yeast (Pan et al., 2006; Tong et al., 2004) with mutational spectrum o f colorectal cancers found i n studies like the one presented here may help to highlight potential targets for this therapeutic strategy. Indeed, mutations o f 4 non-essential cohesion genes [ctf4A, ctf8A, ctfl8A,  and dcclA) are  synthetically lethal with mutations o f 5 different C I N gene homologues which are mutated in colorectal cancer (Fig. 3.2). 3 o f the 4 same cohesion gene mutations (ctf4A, ctf 18 A, and dcclA) are also synthetically lethal with mutations o f C L N gene homologues which are mutated in other cancer types (Fig 2.9). Such analyses suggested that these common synthetic lethal interactors may be attractive drug targets that are effective to a broad spectrum o f C L N tumors with C I N gene mutations.  90  T a b l e 3.1 R e l a t i o n s h i p o f 100 h u m a n candidate C I N genes u s e d i n ( W a n g et a l . , 2 0 0 4 b ) w i t h yeast genes  The 100 human candidate CIN genes were searched for the best yeast homologue by BLASTp according to the proteome database (www.proteome.com), and the e-value is indicated. The list is sorted by the human gene name in ascending order. 19 genes contain only hCT# but not Genbank accession #, so the gene identities of these human genes are not known (highlighted in gray). 3 genes (ATR, POLE, and RAD51C) have 2 different hCT#, representing different isoforms (highlighted in orange). 13 human genes have no BLASTp hit in yeast (highlighted in purple, or blue, which have putative homologs with an e-value >lE-3). 66 human genes yield a yeast hit by BLASTp with an e-value <lE-3. The best yeast hits for these 66 genes were reciprocally searched for their best human homologue by BLASTp. Among these, 30 of them were reciprocal best hits (highlighted in yellow). 15 of the yeast best hits were found in the CIN screens described in Chapter 2 (highlighted in red), and 10 of the 15 are reciprocal best hits; 3 of them correspond to family members of yeast DUN1.  91  Table 3.1  Page 1 of 2  No.  Celera accession  Genbank accession  Human Gene  5 90 42 43 1 80 9 99 19 37 46 72 82 21 11 61 54 85 55 81 51 3 31 96 86 16 70 58 35 32 57 98 13 59 65 84 64 77 74 88 91 7 89 34 92 8 63 95 100 68 66 14 73 25 28 49 24 6 71 97 60 40 78 52 62 2 69 50  hCT12678 hCT7133 hCT1826039 hCT1826040 hCT10388 hCT31470 hCT14094 hCT9356 hCT1643963 hCT1816212 hCT18305 hCT30161 hCT32245 hCT1646711 hCT14628 hCT23387 hCT20446 hCT32971 hCT20952 hCT32115 hCT1961597 hCT11790 hCT17786 hCT8974 hCT401149 hCT16364 hCT29475 hCT2308143 hCT1788172 hCT1783089 hCT22552 hCT9098 hCT14856 hCT2334792 hCT24254 hCT32914 hCT23665 hCT30866 hCT30362 hCT6664 hCT7448 hCT13660 hCT7084 hCT1787138 hCT7976 hCT14027 hCT23655 hCT87415 hCT9836 hCT28965 hCT28290 hCT15239 hCT30207 hCT1686635 hCT1767458 hCT18816 hCT1686440 hCT13183 hCT29790 hCT9089 hCT23382 hCT1823014 hCT30904 hCT19876 hCT23494 hCT11285 hCT29050 hCT18916  NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM  005883 014840 001184 001184 016374 006768 020439 172080 001254 001813 022909 001274 007194 001340 001348 004734 015070 007068 000123 004111 017975 012415 014635  NM NM NM NM  014586 014915 032430 014708  NM NM NM NM NM NM NM NM NM NM  003550 014791 152619 016195 005591 002485 021076 004153 177990 002592  NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM  002646 002647 006219 005026 002649 002691 006231 006231 006904 133377 133338 005732 133487 058216 058216 133627 134422 003579 002913 002914 002915 002916 007370 002945 012238  APCL APC2 ARK5 ATR . ATR BCAA ARID4B; BRCAA1 BRAP CAMK1G VWS1 CAMK2B CDC6 CENPE CENPH CHK1 CHK2 CYLC2 DAPK3 DCAMKL1 DING DMC1 ERCC5 RAD27 FEN1 FLJ10036 FSBP RAD54B GCC185 HCA127 HUNK KIAA1074 KIAA1811 BRSK1 KNTC1 ROD LATS1 MAD1L1 MELK MGC45428 MPH0SPH1 MRE11A MRE11 NBS1 NEFH 0RC1L PAK7 PCNA PIK3C2A PIK3C2B VPS34 PIK3C3 PIK3CB PIK3CD PIK3CG P0LD1 ROLE_.ROLE . . . . . . PRKDC RAD1 RAD17 RAD50 RAD51 RAD51C " RAD51C RAD51L3 RAD52 RAD54L RFC1 RFC2 RFC3 RFC4' RFC5 RPA1 SIRT1 SIRT2 SIRT3 SIRT4  NM 012239 NM 176827  Other gene name  92  Top yeast hit E-value 5E-04 VAC8 SNF1 2E-60 1E-108 MEC1 1E-108 MEC1 N/A 7E-54 YHL010C CMK2 1E-59 CMK2 1E-52 8E-33 CDC6 2E-54 KIP3 N / A * .*.. . . CHK1 9E-45 DUN1 3E-50 YFR016C 4E-09 CMK1 1E-48 DUN1 2E-53 N/A. . PDS1 ? 1E-99 DMC1 RAD2 7E-47 RAD27 1E-104 N/A RDH54 1E-122 NUM1/PAC12 9E-08 N/Aft-K'./:^ • SNF1 1E-51 AKR2 9E-09 SNF1 2E-76 N/A CBK1 1E-100 N/A MAD1 ? SNF1 2E-65 DUN1 4E-52 6E-37 CIN8 1E-104 MRE11 N/A XRS2 ? 6E-13 CHS5 ORC1 2E-39 STE20 5E-80 1E-52 POL30 VPS34 3E-48 VPS34 3E-48 VPS34 1E-139 VPS34 2E-47 2E-57 VPS34 VPS34 7E-51 CDC2 0E+00 POL2 0E+00 POL2 0E+00 1E-34 TOR1 N/At^.-.:„,.; RAD24 4E-17 2E-59 RAD50 RAD51 1E-112 3E-18 DMC1 DMC1 3E-18 DMC1 1E-13 RAD52 5E-41 RAD54 1E-160 1E-115 RFC1 RFC4 1E-109 2E-67 RFC5 RFC3 5E-42 5E-80 RFC3 RFA1 1E-92 SIR2 1E-56 HST2 2E-59 4E-60 HST2 7E-14 HST2  Yeast hit found Reciprocal top E-value human hit in CIN screens? ARMC3 PRKAA2 ATR ATR  1E-23 VAC8 1E-113 1E-108 1E-108  BRAP CMK1D CMK1D CDC6 KIF18A  7E-54 1E-60 1E-60 8E-33 1E-81  CHEK1 DCAMKL1 NEF2 CMK1D DCAMKL1  9E-45 2E-53 1E-10 9E-63 2E-53  DMC1 ERCC5 FEN1  1E-99 7E-47 1E-104 RAD27  RAD54B RSN  1E-122 RDH54 3E-09  PRKAA2 ZDHHC17 PRKAA2  1E-113 1E-48 1E-113  STK38I  1E-131  PRKAA2 DCAMKL1 KIF11 MRE11  MAD1 1E-113 2E-53 DUN1 ....... 1E-48 CIN8 1E-104 MRE11 ..  NEFH ORCL1 PAK1 PCNA PIK3C3 PIK3C3 PIK3C3 PIK3C3 PIK3C3 PIK3C3 POLD1 POLE POLE FRAP1  6E-13 2E-39 1E-111 1E-52 1E-139 1E-139 1E-139 1E-139 1E-139 1E-139 0E+00 0E+00 0E+00 0E+00  CDC6 -  V  DUN1 DUN1  _ .  :  RAD17 RAD50 RAD51 DMC1 DMC1 DMC1 RAD52 RAD54L RFC1 RFC2 RFC3 ' RFC5 RFC5 RPA1 • SIRT1 SIRT3 SIRT3 SIRT3  4E-17 2E-59 1E-112 1E-99 1E-99 1E-99 5E-41 1Er160 1E-115 1E-109 2E-67 5E-80 5E-80 . 1E-92 1E-56 4E-60 4E-60 4E-60  RAD24 RAD50 RAD51  RAD52 RAD54  1 •' ..  Table 3.1 67 12 33 56 36 87 83 48 75 23 76 79 39 4 10 15 17 18 20 22 26 27 29 30 38 41 44 45 47 53 93 94  hCT28652 hCT14647 hCT1786284 hCT21449 hCT17934 hCT6634 hCT32452 hCT18373 hCT30596 hCT16627 hCT30844 hCT31391 hCT1817729 hCT12352 hCT14327 HCT15320 hCT1642589 KCT1643619 hCT1644019 hCT1657158 hCT173001 hCT1766645 hCT1770914 hCT1775724 hCT1817706 hCT1824077 hCT1829493 hCT1829782 hCT1834200 hCT201497 hCT87379 hCT87385  NM 012241 NM 016539 NM 016538 NM 018225 AA447812 NM 007027 NM 003292 NM 004628 NM 006297 NM 005432 NM 004724 NM 007057 NM 012291  SIRT5 SIRT6 SIRT7 SMU-1 SNRK T0PBP1 TPR XPC XRCC1 XRCC3 ZW10 ZWINT  Page 2 of 2  SIR2 HST2 HST1 PFS2 SNF1 N/A AGA1 RAD4 N/A DMC1 N/A , N/A ESPL1/SEPARASE ESP1  93  9E-16 3E-12 3E-10 7E-17 3E-53  SIRT1 SIRT3 SIRT1 WRD33 PRKAA2  1E-56 4E-60 5E-48 9E-70 1E-113  2E-04 3E-26  MUC17 XPC  3E-25 3E-26  2E-19  DMC1  1E-99  1E-36  ESPL1  1E-36  .  T a b l e 3.2 List o f yeast strains used i n Chapter 3 Strain Genotype YPH986 MATa/MATaura3-52/ura3-52 trplA-63/trplA-63 his3A-200/his3A-200 leu2A-l/leu2A-l ade2-101/ade2-101 Iys2-801/lys2-801 CFIII(CEN3.L)-HIS3 SUP 11 YKY1038 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 smcl-Q449W::kanMX CFIII(CEN3.L)-HIS3 SUP 11 YKY1042 MATaura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 smcl-Q449W::kanMX CFIII(CEN3.L)-HIS3 SUP 11 YKY1053 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 smcl-L574M::kanMX CFIII(CEN3.L)-HIS3 SUP 11 YKY1051 MATaura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 smcl-L574M::kanMX CFIII(CEN3.L)-HIS3 SUP 11 YKY1034 MATaura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 smcl- VI 187I::kanMX CFIII(CEN3.L)-HIS3 SUP 11 YKY1031 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 smcl- VI 1871::kanMX CF11I(CEN3.L)-HIS3 SUP 11 YKY1011 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 SMCl::kanMX CFIII(CEN3.L)-HIS3 SUP 11 YKY1023 MATaura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 SMClr.kanMX CFIII(CEN3.L)-HIS3 SUP 11  94  T a b l e 3.3 101 candidate C I N genes a n a l y z e d i n this study  88 human genes were selected from the non-essential yeast CEN genes based on the extent of yeast/human similarity (63 had a e-value <1E-10) and CIN phenotype strength in yeast. 2 human genes were selected based on their similarity to 2 non-essential CTF genes identified by traditional random mutagenesis (Spencer et al., 1990) but missed in the high-throughput screens. Another 11 human genes were selected because they have sequence similarity to essential yeast CTF genes identified by traditional random mutagenesis (Spencer et al., 1990). Most of these human genes correspond to the top hits of yeast CIN genes, except for 5 human genes which are second or third hits (families are indicated in orange). In total, 101 candidates were analyzed, 74 of which had a e-value <1E-10. The list is sorted in ascending order by the yeast gene name. 15 candidate genes are involved in cohesion (indicated in yellow).  95  Table 3.3 No. Yeast O R F Yeast gene 1 YAR015W  ADE1  2 YGL234W  ADE5.7  3 YGR061C  ADE6  4 YBR231C  AOR1  5 YLR085C  ARP6  6 YJL115W  ASF1  7 YER016W  BIM1/CTFS3  8 YDL074C  BRE1  9 YOR026W  BUB3  Essential?  ctf  0 0 0 4 0 3 wrong 3 5 3 0 0 5 2 3 #N/A 1 0 3 3 3 5 3 5 . 3 wrong 4 3 0 3 3 5 3 0 3 4 3 5 3 6 3 wrong 0 5 0 0 3 nd 3 #N/A #N/A 3 1 4 wronq 5 0 contam 0 4. 4 0 contam 3 3 nd . 0 4 . 1 2 2 . 3 3 4. . 3 ... 5 0 0 0 3. 0 wrong 0 4 . . , 3 •' #N/A 0 4 wrong wrong wrong 2 •' 0 2 1 2 3 #N/A 3 0 0 4 0 contam 0 3 wrong 5 0 4 0 2 wrong 4 5 2 0 4 0 5 0 4 1 nd 3 4 0 3 0 3 0 2 wrong 0 0 0  10 YJL194W  CDC6/CTF10  11 YLR418C  CDC73  12 YGL003C  CDH1  13 YPL008W  CHL1/CTF1  14 YPL008W  CHL1/CTF1  15 YMR198W  CIK1  16 YEL061C  CIN8  17 YPR120C  CLB5  18 YMR048W  CSM3  19 YMR078C  C T F 18  20 YLR381W  CTF3  21 YPR135W  CTF4  22 YHR191C  CTF8  23 YJL006C  CTK2  24 YCL016C  DCC1  25 YIR004W  DJP1  26 YGL240W  DOC1  27 YFR027W  EC01/CTF7  Ess  28 YFR027W  EC01/CTF7  Ess  29 YOR144C  ELG1  30 YBR026C  ETR1  31 YEL003W  GIM4  32 YCR065W  HCM1  33 YBR009C  HHF1  34 YPR067W  ISA2  35 YPR141C  KAR3  36 YDR532C  KRE28  37 YDR378C  LSM6  38 YJL030W  MAD2  39 YPR046W  MCM16/CTFS155  40 YDR318W  MCM21/CTF5  41 YFL016C  MDJ1  42 YOL064C  MET22  43 YOR241W  MET7  44 YDR386W  MUS81  45 YBL079W  NUP170/CTFS141  46 YDL116W  NUP84  47 YKL055C  OAR1  48 YKR087C  OMA1  49 YGR078C  PAC10  50 YHR064C  PDR13  51 YMR076C  PDS5/CTF11  52 YLR273C  PIG1  53 YOL054W  PSH1  54 YPL022W  RAD1  55 YML095C  RADIO  56 YCR066W  RAD18  57 YER173W  RAD24  58 YLR032W  RAD5  59 YER095W  RAD51  60 YML032C  RAD52  61 YDR076W  RAD55  62 YDR004W  RAD57  63 YDL059C  RAD59  64 YGL058W  RAD6  65 YDR014W  RAD61/CTF6  66 YDR217C  RAD9  67 YNL072W  RNH35  68 YJR063W  RPA12  Ess  Ess  bim  Page 1 of 2 Protein Accession  mRNA Accession  phosphoribosylaminoimidazole c 6E-08  NP 006443  NM 006452.2  phosphoribosylglycinamide form 0E+00  NP 000810  NM 000819.3  phosphoribosytformylQlycinamidi 1E-176  NP 036525  NM 012393.1  craniofacial development proteir 5E-10  NP 006315  NM 006324.1  0  ARP6 actin-related protein 6 hor 2E-44  NP 071941  NM 022496.2  22 16 11 24 #N/A 2 0 7 . 7 0 0 4 12 29 0 27 18 contam 18 13 3 #N/A #N/A 12. 5 19 2 6 3 40 30 0 0 4 , 3 19 . wrong 13 .'. 9 #N/A contam  ASF1 anti-silencing function 1 h<8E-51  NP 054753  NM 014034.1  microtubule-associated protein. 2E-28  NP 036457  NM 012325.1  RPN20, ring finger protein 20; he5E-26  NP 062538  NM 019592.5  BUB3 budding uninhibited bv be 2E-24  NP 004716  NM 004725.1  CDC6 homolog; CDC18 (ceil div 2E-32  NP 001245  NM 001254.2  parafibromin; chromosome 1 op<9E-12  NP 078805  NM 024529.3  Fzr1 protein; fizzy-related proteii 1E-92  NP 057347  NM 016263.2  OEAD/H (Asp-Glu-Ala-Asp/His) 1E-112  NP 085911  NM 030653.2  CHLR2/DDX12, DEAD box prott4E-94  AAB06963.1  U33834  golgi autoantigen, golgin subfarr 4E-07  NP 002069  NM 002078.3  kinesin family member 11; Eg5; 2E-67  NP 004514  NM 004523.2  evelin B1; G2/mitotic-specific cy( 6E-39  NP 114172  NM 031966.2  timeless-interacting protein; tipin 8E-08  NP 060328  NM 017858.1  CTF18, chromosome transmissi 3E-36  NP 071375  NM 022092.1  LRPR1 (CENPI), follicle-stimulat see Measday V, 20C NP 006724  NM 008733.2  WD repeat and HMG-box DNA t 1E-18  NM 007086.1  alf  Top human hit  31 12 16 0  .  E-value  NP 009017  hCTF8, hypothetical protein M G see Mayer M, 2001 NP 00103523 NM 001039690 2E-11  NP 003849  NM 003858.2  hypothetical protein MGC5528 [I 8E-11  NP 076999  NM 024094.1  DnaJ (Hsp40) homolog, subfam 4E-18  NP 061854  NM 018981.1  anaphase-promoting complex si 5E-22  NP 055700  NM 014885.1  establishment factor-like protein 5E-11  NP 443143  NM 052911.1  E S C 0 2 , Establishment of cohes  NP 001017420.1  NM_001017420  hypothetical protein FLJ12735 [f 2E-05  NP 079133  NM 024857.3  nuclear receptor-binding factor 1 5E-47  NP 057095  NM 016011.1  1E-15  NP 036526  NM 012394.2  forkhead box 12; Blepharophimc 1E-20  NP 075555  NM 023067.2  histone 2, H4; H4 histone, family 2E-37  NP 003539  NM 003548.2  HESB like domain containing 1 [ 2E-05  NP 919255  NM 194279.1  kinesin family member C1 (Hom< 2E-69  XP 371813  XM 371813.1  retinobiastoma-binding protein 1 3E-06  NP 002883  NM 002892.2  5E-09  NP 009011  NM 007080.1  MAD2-like 1; MAD2 (mitotic arre 5E-38  NP 002349  NM 002358.2  high density lipoprotein binding r. 2E-03  NP 976221  NM 203346.1  SCC1/MCD1, RAD21 homolog; 3E-02  NP 006256  NM 006265.1  DnaJ (Hsp40) homolog, subfam 2E-31  NP 005138  NM 005147.3  inositoKmyoM (or 4)-monophosr 4E-05  NP 005527  NM 005536.2  folylpolyglutamate synthase; foly 1E-11  NP 004948  NM 004957.2  MUS81 endonuclease homolog 1E-18  NP 079404  NM 025128.3  nucleoporin 155kDa isoform 1; r 2E-30  NP 705618  NM 153485.1  cyclin K (Homo sapiens]  prefoldin 2 [Homo sapiens]  Sm protein F (Homo sapiens]  3E-07  nuclear pore complex protein [H 5E-16  NP 065134  NM 020401.1  .  DKFZP566O084 protein [Homo 2E-13  NP 056325  NM 015510.3  30 3 3' • #N/A 0 11 5 2 19 6 5 7 22 18 15 2 25 5 10 6 5  metalloprotease related protein 6E-29  NP 660286  NM 145243.2  von Hippel-Lindau binding prote 4E-30  NP 003363  NM 003372.3  heat shock 70kDa protein 8 isofc 2E-56  NP 006588  NM 006597.3  androgen-induced prostate proli 1E-29  NP 055847  NM 015032.1  protein phosphatase 1, regulato 2E-06  NP 005389  NM 005398.3  tripartite motif-containing 25; Zin 1E-07  NP 005073  NM 005082.3  excision repair cross-compleme 1E-109  NP 005227  NM 005236.1  excision repair cross-compleme 1E-12  NP 001974  NM 001983.2  postreplication repair protein hR, 9E-20  NP 064550  NM 020165.2  RAD17 homolog isoform 2; Rad 8E-17  NP 579917  NM 133339.1  SWI/SNF-related matrix-associa 5E-70  NP 620636  NM 139048.1  RAD51 homolog protein isoform 1E-122  NP 002866  NM 002875.2  RAD52 homolog isoform alpha; 2E-40  NP 002870  NM 002879.2  RAD51-like 3 isoform 1; recomb 1E-05  NP 002869  NM 002878.2  RAD51-like 1 isoform 3; RecA-li 4E-19  NP 598193  NM 133509.2  RAD52 homolog isoform alpha; 8E-09  NP 002870  NM 002879.2  ubiquitin-conjugating enzyme E2 7E-61  NP 003327  NM 003336.2  hypothetical protein LOC57821 4E-03  NP 067002  NM 021179.1  dentin sialophosphoprotein prep 1E-07  NP 055023  NM 014208.1  ribonuclease HI, large subunit [H 7E-46  NP 006388  NM 006397.2  zinc ribbon domain containing. 1 1E-14  NP 740753  NM 170783.1  B  ...  96  Table 3.3 69 YJL140W  RPB4/CTF15  70 YIL018W  RPL2B  71 YDL204W  RTN2  72 YOR014W  RTS1  73 YJL047C  RTT101  74 YDR289C  RTT103  75 YDR159W  SAC3  76 YDR180W  SCC2/CTF12/CHL8  77 YIL026C  SCC3/IRR1/CTFs16! Ess  78 YIL026C  SCC37IRR1/CTFS163 Ess  79 YIL026C  SCC3/lRR1/CTFs16d Ess  60 YMR190C  SGS1  81 YLR058C  SHM2  82 YBL058W  SHP1  83 YLR079W  S1C1/CTFS127  84 YER116C  SLX8  85 YFL008W  SMC1/CTFS166.  86 YFL008W  SMC1/CTFS166  Ess  87 YJL074C  SMC3  Ess  88 YOR308C  SNU66  89 YGR063C  SPT4/CTFS138  90 YPR032W  SR07  91 YBR112C  SSN6/CYC8  92 YOL072W  THP1  93 YNL273W  TOF1  94 YLR234W  TOP3  95 YAL016W  TPD3  96 YML028W  TSA1  97 YGR184C  UBR1  98 YDL156W  YDL156W  99 YLR193C  YLR193C  100 YGR270W  YTA7  101 YGR285C  ZU01  .  Ess  Ess  3 wronq 0 wronq wronq 2 contam 3• 3 3 ., 3 0 0 2 3 1 ' , 3 3 #N/A 2 1 wronq 0 wronq 3 0 nd 0 3. . 0 0 wi-ohq 0  #N/A 0 0 0 wronci 0 wronq #N/A #N/A #N/A #N/A 4. 4 4 6 . 5 #N/A #N/A #N/A 0 0 2 nd 4 5 5 nd 4 3 2 2 4 4 :  Page 2 of 2  #N/A 17 1619 15. 0 5 #N/A #N/A #N/A #N/A 14 . 2 2 31 wronq . #N/A #N/A #N/A 0 0 3 80 19 10 42 75 8 0 2 2 6 wronq  DNA directed RNA polymerase I 3E-12  NP 004796  NM 004805.2  ribosomal protein L6; 60S ribosc 1E-102  NP 150644  NM 033301.1  reticulon 2 isoform A: NSP-like p 5E-08  NP 005610  NM 005619.3  delta isoform of regulatory subur 1E-148  NP 006236  NM 006245.2  9E-06  NP 003582  NM 003591.2  chromosome 20 open reading fr 3E-14  NP 067038  NM 021215.2  minichromosome maintenance c 4E-26  NP 003897  NM 003906.3  NIPBL. IDN3 protein isoform A [I 3E-19  NP 597677  NM 015384.3  STAG1, stromal antigen 1; nuclc 2E-21  NP 005853  STAG3, Stromal antigen 3 (stror 3E-13  NP 038579.2  NM 005862.1 NMJM2447  STAG2, Stromal antigen 2. a me 2E-11  NP 006594.3  NM_O066O3  Bloom syndrome protein [Homo 1E-115  NP 000048  NM 000057.1  serine hydroxymethyltransferasr 1E-148  NP 004160  NM 004169.3  p47 protein isoform a THorno sat 6E-34  NP 057227  NM 016143.3  hypothetical gene supported bv 7E-02  NP 963859  NM 201565.1  ring finger protein 10 [Homo sap 7E-08  NP 055683  NM 014868.3  SMC1 structural maintenance of 1E-149  NP 006297  SMC1L2, Protein with strong sirr SE-41  NP 683515.3  NM 006306.2 NM_148674  CSPG6, Chondroitin sulfate prot 1E-45  NP 005436.1  NM_0O5445.3  squamous cell carcinoma antige 2E-07  NP 005137  NM 005146.3  suppressor of Ty 4 homolog 1 rh 1E-18 4E-14 tomosvn-like [Homo sapiensl  NP 003159  NM 003168.1  X P 045911  XM 045911.8  cullin 2 [Homo sapiens]  1  UTX, ubiquitously transcribed te 9E-44  NP 066963  NM 021140.1  hypothetical protein FLJ11305 [>• 6E-07  NP 060856  NM 018386.1  timeless homolog [Homo sapien 5E-07  NP 003911  NM 003920.1  topoisomerase (DNA) III alpha; t 1E-124  NP 004609  NM 004618.2  beta isoform of regulatory subun 1E-133  NP 859050  NM 181699.1  peroxiredoxin 2 isoform a; thiore 7E-71  NP 005800  NM 005809.4  ubiquitin ligase E3 alpha-ll; likely 9E-28  NP 056070  NM 015255.1  hypothetical protein FLJ12973 [h 3E-19  NP 079184  NM 024908.1  similar to Px19-like protein (25 k 2E-22  XP 371496  XM 371496.2  two AAA domain containing prot 1E-131  NP 054828  NM 014109.2  similar to M-phase phosphoproK 1E-48  X P 379909  XM 379909.1  97  T a b l e 3.4 S o m a t i c mutations identified i n candidate C I N genes i n C I N colorectal cancer c e l l s 20 mutations were identified in 8 genes. The number of mutations found and the number of tumors sequenced are indicated. The nucleotide mutation and corresponding amino acid mutation are indicated by the position based on the coding sequence (CDS), followed by the wild-type sequence>tumor sequence. Thus, 2 different nucleotides in the tumor sequence represent a heterozygous mutation, and a single nucleotide in the tumor sequence represents a homozygous or hemizygous mutation. The corresponding amino acid change is indicated in the same manner. The corresponding tumor, exon number and primer used are indicated. The yeast gene used as query and the e-value are also indicated.  Human . No. tumors Human Protein No; Yeast Gene e-value • Human RNA ID Gene Gene ID mutations sequenced Name No. Name 5 132 NM 006306.2 NP 006297.2 1E-149 1 SMC1L1 SMC1  2 CSPG6  SMG3  1E-45  NM 005445.3  NP 005436.1  130  5 •  *.  .  PRIMER  Exon  Somatic Mutation  hCT9553-7 hCT9553-8 hCT9553-10 hCT9553-16 hCT9553-24 CSPG6 23 . f'/.-,';C.SPGfr7/, i CSPG6 8 r AeSPG6l:12., ' r 1 CSPG6_21 hCT2293447 9 3 hCT2293447 9 4 hCT2293447 10 1 YC03C04F hCT2293447 40 STAG2 24 STAG3 32 YC08C06B YC14C06D YC16C06G  7 8 10 16 24 23  1186T>CT: 396F>UF 1300C>T: 434R>W 1680C>CG: 560I>I/M 2562 2563het insA 3556G>AG: 1186V>IA/ 2635C>CT:879R>R/X 415G>AG:139V>IA/ 512G>AGi171R>Q/R 100C>CT:334L>L/F 2321G>A:774R>K 1435C>CT:479R>R/X 2967 2968het insT 1660C>CT:554Q>Q/X 5378T>TA: 1793M>M/K 6893G>AG: 2298R>H/R 2456C>CT:819S>S/F 33963G>AG 3128C>AC: 1043A>D/A 2380A>AC:794Y>Y/S 370C>CT:124R>R/X  4  :  3 NIPBL  SCC2  3E-19  NM 015384.3  NP 597677.2  5  132  4 STAG2 5 STAG3 6 BLM 7 UTX 8 RNF20  SCC3 SCC3 SGS1 SSN6/CYC8 BRE1"  2E-11 3E-13 1E-115 9E-44 5E-26  NM 006603 NM 012447.2 NM 000057.1 NM 021140 NM 019592  NP NP NP NP NP  1 1 1 1 1  34 34 132 36 36-  006594.3 036579.2 000048.1' 066963.1 062538.5  7  8 12 21 ' 8 9 9 28 39 24 31 15 17 3  Tumor  ;  C094 HX8 CX3 HX171 HX129 MX13 HX155 . HX171 HX152 HX133 HX7 C071 HX168 MX24 HX171 HX147 HX110 HX63 HX68 HX88  F i g u r e 3.1 Mutations i n human SMC1L1 in colorectal cancers and analogous mutations in yeast SMC1 A. Protein sequence alignment of human SMC J LI with yeast SMC] reveals 27.8% identity and 42.1% similarity. Known domains are highlighted: the P-loop containing ATPase domain at N - and C-terminus are in blue; the SMC hinge domain is in orange. Somatic mutations found in human cancers and the analogous yeast amino acids are shown in red boxes. A nucleotide insertion leading to frameshift and truncation is indicated by a red arrow and stop sign. B. The table indicates the corresponding mutations constructed. C. Frequency of chromosome fragment loss per division is indicated for WTmd missense mutant haploids. The lower panel shows the sectoring phenotypes of the VI1871 mutant and control WTcells.  99  F i g u r e 3.1 A. 1  (l (i 1 ) !  teSMClLl  ?i R*|ss  rs;  R  3 7 fJ ? r 3 a -  r i p : 3 ; ; 5 •• y~*i-;: A :  ; rv ~  1  1  E M ;  ; s V7T l § 3 i : |  S  I) : M 5 R i V 5 § J s s r K S Y § o % - r ^ ; r ; » S K F i § : : ; ? % - ; s i . » . s \ ^ ; i ; 3 r v - 5 \ ^ s s i R S S i K S M C I ( 1( )  cow*.*  (!) as  i  : E : NFRSYXS  •62; 82  90  W  110  ,120  J r £ . : | s u | v | x c E N | = : v : 2 i  I S  J30  I K I I I X  ,140  •• E E : . ;  tE--S*ESR?r*M:vj- --«IE'. 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S K I I T  3 1 3Y  3  100  B Human SMC 1 LI amino acid substitution F396L R434W I560M Insertion of A between coding sequence 2562 & 2563, leading to amino acid change starting from 855 and termination at amino acid 864 VI1861  Type of SMC1L1 mutation Homo/Hemi Hetero Hetero Hetero  Yeast SMC1 amino acid substitution L380 (no mutation made) Q449W L574M I877Z V11871  Hetero  c Frequency of half-sectored & red colonies  0.008 0.007 0.006 « 0.005 a 0 . 0 0 4 I" 0 . 0 0 0.002 0.001 0  3  4> Strain (haploid)  7 1*0  V1187I  101  m  • • \ •  »*•  aft*  -  • * * *• ft JfV  F i g u r e 3.2 Synthetic lethal interactions o f yeast C I N genes whose human homologues were found to be mutated in colorectal cancers (which are placed on the r i m and depicted in blue fonts). A. Only genes synthetic lethal with more than 1 of these CIN genes are shown. B. Only genes synthetic lethal with at least 5 CIN genes are shown.  102  CHAPTER 4 Characterization of MMS22, MMS1, RTT101 and RTT107 in the Maintenance of Genome Integrity  103  4.1 I n t r o d u c t i o n  One o f the goals o f performing genome-wide chromosome instability screens is to identify novel genes and characterize their functions. A m o n g the 293 genes identified in the C I N screens described i n Chapter 2, 46 (16%) were uncharacterized (see Appendix 1 and 2; by G O Slim Mapper on S G D , http://db.yeastgenome.org/cgibin/GO/goTermMapper). To prioritize genes for further study, I first examined the 34 genes that were identified i n all 3 CLN screens. M a n y o f the genes within this subset are known to be important for maintaining genome integrity, such as those that function at the kinetochore, in cell cycle checkpoints and in D N A repair pathways. A t the time the screens were completed, only a few o f the 34 genes were largely uncharacterized, including NCE4, MMS1 and MMS22. However, I was able to gain insights into the functions o f these genes by integrating data derived from large scale phenotypic screening (such as the C L N , G C R and drug sensitivity screens (see below)), as well as genetic and physical interaction analyses (Figure 2.5). Mass spectrometry ( M S ) analysis o f immunoprecipitates o f overexpressed tagged Mms22p identified Rttl01p/Cul8p, a cullin, and Rttl07p/Esc4p as interacting proteins (Ho et al., 2002), all o f which were also identified i n the C L N screens. In addition, R t t l O l p and M m s l p / R t t l 0 8 p / K i m 3 p were identified as protein-protein interactors with R o c l p / H r t l p , a R I N G finger protein known to bind to cullins in E3 ubiquitin ligases (Ho et al., 2002). These physical interactions suggested that these proteins may function together to maintain genome integrity. Indeed, RTT (retrotransposition) genes were originally identified in a screen for mutants that increase the transposition rate o f T y l , a long terminal repeat ( L T R ) retrotransposon (Scholes et al., 2001). M a n y o f the rtt mutants, including rttl07A and mmslAlrttl08A,  rttlOlA,  were found to have elevated rates o f gross chromosomal  rearrangement ( G C R ) (Kanellis et al., 2003; Luke et al., 2006; Rouse, 2004). O n the other hand, MMS22 was identified i n another screen set up to look for mutants leading to reduced levels o f T y l retrotransposition, and it was shown that the level o f T y l c D N A was not affected in mms22A mutants, suggesting that Mms22p affects steps that occur  104  after D N A replication, possibly i n the repair o f chromosomal D N A damage at integration sites (Griffith et al., 2003). M a n y C I N mutants are sensitive to D N A damaging agents. Different D N A damaging agents cause different types o f D N A lesions and sometimes one agent generates pleiotropic lesions (Table 4.1 and Figure 4.1). Genes whose proteins function in the same or parallel pathway are expected to display similar genetic interaction profiles and phenotypes such as C L N and drug sensitivity (Parsons et al., 2004; Tong et al., 2004). For example, base excision repair (BER) mutants are sensitive to base damaging agents, but not to U V or LR. Nucleotide excision repair ( N E R ) mutants are sensitive to U V and 4 N Q O , but only moderately sensitive to M M S and LR. Post-replication repair mutants are sensitive to M M S , U V and LR. Homologous recombination ( H R ) mutants are extremely sensitive to IR, but are only moderately sensitive to U V (Figure 4.1). Indeed, MMS genes, including MMS22 and MMS1, were originally identified i n a screen for mutants that are sensitive to the M M S (Prakash and Prakash, 1977). In addition, both mms22A and  mmslA  mutants are sensitive to many other D N A damaging agents including H U , C P T , and moderately sensitive to U V (Baldwin et al., 2005; Bennett et al., 2001; Chang et al., 2002; Hanway et al., 2002; Parsons et al., 2004; Prakash and Prakash, 1977);(Hryciw et al., 2002). Similarly, rttlOlA  and rttlOJA mutants are also sensitive to M M S , H U , and  C P T (Bennett et al., 2001; Chang et al., 2002; Hanway et al., 2002). The drug sensitivity profiles o f these 4 mutants were most similar to that o f post-replication repair gene mutants, but not identical (see below), suggesting they may constitute a novel pathway involved in D N A repair. During the last few years, the role o f Rttl07p, R t t l O l p , M m s l p and Mms22p in D N A damage response has been characterized in more detail as summarized below.  4.1.1  RTT107 R t t l 0 7 p contains 6 B R C T domains, which are usually found i n proteins involved  in signaling, repairing o f D N A damage or cell cycle regulation (e.g. B R C A 1 , X R C C 1 , and 53BP1). rtt!07A causes delays i n S phase, accumulation i n G 2 / M , and an increased  105  fraction o f cells with checkpoint protein Ddc2p foci, which suggest a higher level o f spontaneous D N A damage (Roberts et al., 2006). However, double mutant analysis for M M S sensitivity showed that Rttl07p is not involved i n N E R , H R or cell cycle control (Hanway et a l , 2002). Indeed, Rttl07p is not required at the time o f damage, and rttlOJA mutants are competent for activation o f the intra-S-phase checkpoint, which is indicated by Rad53p phosphorylation. In response to D N A damage occurring i n S phase, R t t l 0 7 p is phosphorylated by the checkpoint protein M e c l p , and R t t l 0 7 p is important for recovery from D N A damage by promoting restart o f stalled replication forks (Rouse, 2004). Slx4p and S l x l p , which form a structure-specific D N A endonuclease required for resolving replication intermediates specifically in the r D N A (Fricke and B r i l l , 2003), physically interact with Rttl07p. The Slx4p-Rttl07p interaction is independent o f D N A damage, and requires the B R C T domains o f Rttl07p. The interaction is required for Meclp-mediated phosphorylation o f Rttl07p (Roberts et al., 2006). In another study, yeast-two-hybrid ( Y 2 H ) analysis o f the N-terminal region o f R t t l 0 7 p (containing 4 B R C T domains) identified Rad55p, Mms22p, T o f l p and S g s l p (Chin et a l , 2006). L i k e the interaction with Slx4p, the physical interaction between R t t l 0 7 p and Rad55p does not depend on D N A damage (Chin et al., 2006). L i k e Rttl07p, Rad55p is also phosphorylated by M e c l p in response to D N A damage, and it forms a heterodimer with Rad57p, which together orchestrates the assembly o f the Rad51p filament on replication protein A (RPA)-coated s s D N A . Taken together, these physical interactions suggest that Rttl07p may associate with s s D N A o f stalled replication forks to modulate repair and reinitiation o f D N A synthesis. Subcellular localization o f Rttl07p is i n agreement with its putative function at stalled replication forks. R t t l 0 7 p displays diffuse nuclear localization i n G I and G 2 / M , which is unaffected by M M S treatment (Chin et al., 2006). However, half o f S phase cells have R t t l 0 7 p foci at the edge o f the nucleus, and treatment with M M S increases the fraction o f cells containing such foci (Chin et al., 2006). Pax2 transactivation domain interacting protein (PTIP), the mammalian protein with highest sequence similarity to  106  Rttl07p, was recently shown to form foci after D N A damage (Manke et al., 2003). The number o f cells with Rttl07p foci also increase in mrclA or toflA mutants, which contain s s D N A accumulated at stalled replication forks (Chin et al., 2006). Interestingly, Rttl07p foci partially overlap with r D N A repeats. These results support that R t t l 0 7 p may bind stalled replication forks that accumulate s s D N A , and may be involved i n the repair o f replication forks that collapse within the r D N A repeats.  4.1.2  RTT101 R t t l O l p is one o f the 3 cullins i n S. cerevisiae, with demonstrable ubiquitin ligase  activity in vitro, but as yet no known substrate in vivo (Michel et al., 2003).  rttlOlA  mutants accumulate with a short spindle and nucleus positioned at the bud neck (Luke et al., 2006; M i c h e l et al., 2003). The anaphase onset in rttlOlA mutants is delayed, and this is dependent on the intra-S-phase checkpoint ( M e c l p and Rad9p) (Luke et al., 2006; M i c h e l et al., 2003). rttlOlA mutants display several phenotypes resembling rttlOlA mutants. For example, rttlOlA mutants have increased numbers o f D d c l p and Rad52p foci, indicating an increase in spontaneous D N A damage (Luke et al., 2006). rttlOlA mutants are also competent in Rad53p checkpoint activation i n response to H U treatment (Luke et al., 2006). Based on double mutant analysis for M M S sensitivity, R t t l O l p is not involved i n non-homologous end joining ( N H E I ) or H R (Luke et al., 2006; M i c h e l et al., 2003). L i k e R t t l 0 7 p , R t t l O l p may play a role i n replication fork reinitiation or progression. In rttlOlA mutants, replication forks arrested at natural pause sites (e.g. r D N A barriers, centromeres) are more unstable, as indicated b y the increased formation of extrachromosomal r D N A circles (Luke et a l , 2006). rttlOlA mutants cannot complete D N A replication during recovery from M M S , which induces fork arrest. O n the other hand, rttlOlA mutants can recover from H U , which causes fork pausing. These results suggest that R t t l O l p promotes fork progression through alkylated D N A (Luke et al., 2006).  107  4.1.3 MMS22  a n d MMS1  Phenotypically, both mms22A and mmslA mutants exhibit slow growth and abnormal cell morphology including large, round and elongated cells (Araki et al., 2003; H r y c i w et a l , 2002). To determine whether MMS22 and MMS1 function i n known repair pathway, double mutant analysis for M M S sensitivity was performed and suggested that both MMS22 and MMS1 are not involved i n N H E J , and N E R (Araki et al., 2003; H r y c i w et al., 2002). However, mms22A is synthetically lethal with rad6A (post-replication repair) and rad52A (homologous recombination repair) (Araki et al., 2003; H r y c i w et al., 2002) , and mmslA is also synthetically lethal with rad52A in some strain backgrounds (Araki et al., 2003; H r y c i w et al., 2002), suggesting that MMS22 and MMS1 may function in a pathway redundant to RAD52 and RAD6. A n mms22A mmslA  double mutant exhibits  M M S , H U , U V sensitivity that is similar to the mms22A mutant, indicating that mms22A is epistatic to mmslA (Araki et al., 2003). mms22A is also epistatic to rttlOlA  and  rttl07A  (Baldwin et al., 2005). Indeed, sensitivity o f mmslA to D N A damaging agents is suppressed by overexpression of MMS22, but not vice versa, suggesting that MMS1 acts upstream o f MMS22 in a novel repair pathway. Besides genetically interacting with D N A repair genes, MMS22 and MMS1 also genetically and physically interacts with some essential genes involved i n replication initiation. First, high-throughput Y 2 H studies showed that M m s 2 2 p (as prey) interacts with P s f l p and Psf2p, 2 o f the 4 essential subunits o f the G I N S complex (Hazbun et al., 2003) . The G I N S complex binds to D N A replication origins and facilitates assembly o f the D N A replication machinery (Hazbun et al., 2003; Takayama et al., 2003). Second, mutations in MMS22 and MMS1 were both identified in a screen for mutations synthetically lethal with mcml0-l  (Araki et al., 2003). M c m l 0 p / D n a 4 3 p is essential for  replication initiation and the disassembly o f pre-replication complex (pre-RC) after initiation (Araki et al., 2003). Therefore, M c m l O p is required for the smooth passage o f replication forks through obstacles such as those created by pre-RCs assembled at active or inactive replication origins. mcml0-l  causes replication fork pausing at active and  silent origins (Araki et al., 2003). Interestingly, mcml0-l,  108  mms2A and mmslA  are all  synthetically lethal with dna2-2 (Budd et al., 2005). DNA2 encodes an essential D N A replication protein that contains helicase and single-stranded nuclease activities, and is involved in the processing o f Okazaki fragments and i n D N A repair. Collectively, MMS22 and MMS1 may constitute a novel D N A repair pathway that is specific for replication-dependent D N A damage (Araki et al., 2003), i n agreement with R t t l 0 7 p and R t t l O l p having roles in the restart o f replication upon D S B s . To further elucidate the role o f MMS2'2 i n D S B repair, a series o f phenotypic, genetic and physical analyses were performed. The survival rate after exposure to a D S B and the kinetics o f D S B repair were determined i n mms22A mutants and compared to wild-type and known D S B repair mutants. Global identification o f Mms22p, R t t l O l p and Rttl07p physical interactors was performed by mass spectrometry ( M S ) and yeast-twohybrid ( Y 2 H ) analyses. Since physical interactors o f R t t l O l p are potential substrates o f this putative ubiquitin ligase, the protein expression levels o f some interactors were tested.  109  4.2 Materials and Methods 4.2.1 Yeast strains and media Yeast strains used i n this study are listed in Table 4.1. M e d i a for growth and sporulation were described previously in Rose et al., 1990. Epitope tagging and gene deletions were made directly at the endogenous loci (Longtine et al., 1998). Yeast transformations were performed as i n (Gietz et al., 1995).  4.2.2 Quantification of chromosome transmissionfidelity(ctf) Quantification o f the ctf phenotype was performed i n homozygous diploid strains containing a chromosome fragment (CF) as in (Shero et al., 1991). Briefly, diploid cells with one C F form pink colonies. D i p l o i d cells that lose the C F form red colonies, whereas those that contain 2 C F s generate white colonies. Chromosome missegregation in the first cell division after plating generates a half-sectored colony, and the frequencies of half-sectored colonies reflect the rates o f chromosome loss and non-disjunction (Shero et a l , 1991).  4.2.3 Genome-wide yeast-two-hybrid screens MMS22, RTT101 and RTT107 were cloned into p O B D 2 as described i n (Cagney et al., 2000). The M m s 2 2 p - G a l 4 p - D N A binding domain fusion protein was functional as determined by rescuing sensitivity o f mms22A to 0 . 2 M H U , 10p:g/ml camptothecin and 0.01% M M S (data not shown). Genome-wide two-hybrid screens were performed as described in (Uetz et al., 2000). Briefly, each screen was performed i n duplicate, and positives that were identified twice were put into a mini-array for retest. Some reproducible positives were observed in many different screens with baits o f unrelated function. These were considered common false positives and were excluded from further analyses.  4.2.4 Co-immunoprecipitation Co-immunoprecipitations were performed as described in (Measday et al., 2002). In brief, yeast extracts were generated using glass beads lysis. The protein concentration  110  of extracts was measured by Bradford assay, and equal amounts (2-5 mg) o f extracts were incubated with a n t i - M Y C - or anti-HA- conjugated beads (Covance) for - 2 4 hrs at 4°C. Beads were washed i n extract buffer for a minimum o f 3 times, and immunoprecipitates were eluted with S D S - P A G E loading buffer.  4.2.5 Mass spectrometry Protein eluates from immunoprecipitation were diluted i n 20 m M Tris p H 8.3, 5 m M E D T A so that the final S D S concentration was no greater than 0.05%. 20 ng/ul o f sequencing-grade trypsin (Fisher) was added, and digestion was allowed to proceed at 37°C overnight. Samples were then purified using C18 ZipTips (Millipore) according to the manufacturer's instructions. One-dimensional reversed-phase liquid chromatography with on-line mass spectrometry on an ion trap mass spectrometer (Model L C Q , ThermoElectron, San Jose, C A ) was performed as described in (Lee et al., 2004), employing a 90 min. binary gradient from 5%-80% solvent B during which each mass spectrum ( M S ) scan was followed by three M S / M S scans. Experimental mass spectra were compared with theoretical spectra generated from sequences from the Saccharomyces  cerevisiae genome by using the S E Q U E S T algorithm (Yates et al., 1995).  Data were displayed and filtered by using the I N T E R A C T software (Han et al., 2001).  4.2.6 Survival assay in HO-induced double strand breaks pJH132 ( p G A L - H O - T R P l ) (Lisby et a l , 2001) was transformed into wild-type, mms22A and rad52A strains. p R S 4 1 4 - T R P l was used as an empty vector control. Equal amounts o f cells were plated on S C - T R P (Galactose) and S C - T R P (Glucose). Survival rate was calculated by the number o f colonies formed on S C - T R P (Galactose) over that formed on S C - T R P (Glucose) after 8 days.  4.2.7 Microscopy Strains used for microscopy were grown i n F P M (Synthetic complete medium supplemented with adenine and 6.5 g/L sodium citrate) i n order to reduce auto-  111  fluorescence. D A P I (300 ng/ml) was added to live cells for visualization o f D N A as described previously (Connelly and Hieter, 1996). Stacks o f microscopy images were taken with a Zeiss A x i o p l a n II operated with Metamorph software (Universal Imaging). The presence o f Rad52p foci was examined i n WT ( Y K Y 8 0 7 ) and mms22A ( Y T K 1 3 6 4 ) strains as described in (Lisby et al., 2003).  112  4.3 Results 4.3.1 mms22A, mmslA, rttlOlA and rtt!07A exhibit sensitivity to DNA damaging agents and chromosome instability I analyzed the genome-wide drug sensitivity screen results b y 2-dimensional hierarchical clustering, and overlaid them with the C I N screen results (Bennett et al., 2001; Birrell et al., 2001; Chang et al., 2002; Giaever et al., 1999; Hanway et al., 2002; Parsons et al., 2004) (Figure 4.2). Two clusters are enriched for C L N mutants; the cluster showing sensitivity to benomyl is enriched for genes functioning at the kinetochore and spindle, whereas the cluster exhibiting sensitivity to D N A damaging agents is enriched for genes involved i n D N A repair and D N A damage checkpoints, including MMS22, MMS I, RTT101 and RTT107. Interestingly, these 4 genes cluster with the M R X  (MRE11,  RAD50, and XRS2) complex, which is involved i n D S B repair, sister chromatid cohesion and telomere maintenance. Both mms22A and mmslA were identified i n all 3 C L N screens, whereas  rttlOlA  and rttl07A were identified i n at least 1 C I N screen (see Chapter 2). In addition, the G C R rates o f the rtt mutants are elevated (Kanellis et al., 2003; Luke et al., 2006; Rouse, 2004). When the frequency o f chromosome transmission fidelity (ctf) was quantified b y half-sectoring assay (Shero et al., 1991), all these mutants exhibited C I N at various levels (Table 4.3).  4.3.2 mms22A exhibits defects in cell cycle progression Based on the C L N phenotype o f mms2'2'A, Mms22p may function i n a cell cycle step that is crucial for chromosome integrity, or it may be important for repairing certain D N A lesions. Indeed, mms22A cells accumulate at G 2 / M phase (Bennett et al., 2001) and display increased cell size (Jorgensen et al., 2002). The absence o f Mms22p could result in a delay i n certain steps o f the cell cycle, or it could induce spontaneous D N A damage, which would activate the cell cycle checkpoint and lead to cell cycle arrest. Fluorescence activated cell sorting ( F A C S ) analysis o f logarithmically growing mms22A cells, compared to w i l d type cells, revealed a larger 2 N peak, which is consistent with  113  abnormalities i n cell cycle regulation and aneuploidy (Figure 4.3A). Budding index analysis also revealed that an increased proportion o f mms22A cells exhibit large buds and D N A in the bud necks. To investigate whether the G 2 / M accumulation is caused by checkpoint activation, mms22A was combined with several checkpoint mutations. F A C S analysis o f these double mutants showed that this G 2 / M delay is not dependent on the spindle checkpoint (mad2A), the D N A damage checkpoint (rad9A, mec3A), or the replication checkpoint (mrclA) (Figure 4.3A). These results suggest that mms22A cells might be slow to enter or exit mitosis, leading to the G 2 / M accumulation. Indeed, mms22A is characterized by slow growth (Araki et a l , 2003). J^Tand mms22A cells were synchronized by arresting them in G l with cc-factor and their cell cycle were followed after release from the block (Figure 4.3C). mms22A cells appear to enter S phase and finish D N A replication at similar times compared to WT; however, mms22A cells enter the next G l at a much later time than WT cells.  4.3.3 mms22A has reduced survival rate with the introduction of DSBs mms22A cells are sensitive to several D N A damaging agents that cause D S B s . To directly test whether mms22A is impaired in D S B repair, the survival rate o f mms22A cells was monitored following the introduction o f an H O endonuclease-induced D S B (Lisby et al., 2001) (Figure 4.4A). mms22A mutants have a lower survival rate (30-50%) than WT (80-90%), but are not completely inviable as observed i n rad52A mutants. Consistent with the reduced survival rate, when the size o f the colonies i n the presence o f a D S B was examined, mms22A cells formed much smaller colonies compared to WT (Figure 4.4B). This result is consistent with a lower sensitivity o f mms22A mutants to M M S compared to rad52A mutants, suggesting a less important role for M m s 2 2 p in D S B repair, compared to Rad52p. It is possible that Mms22p is only responsible for a subset o f D S B s , such as D S B s that occur during replication, or that the M m s 2 2 p pathway is not the major repair route chosen by the cells.  114  Another way to monitor D S B repair is to examine the formation o f D N A repair centres in cells, i n which Rad52p aggregates multiple D S B sites together and recruits other homologous recombination proteins for repair in S and G 2 phases (Lisby et al., 2003). The number o f Rad52p foci is not directly proportional to the number o f D S B s , suggesting that each focus likely represents the repair centre o f multiple D N A lesions (Lisby et al., 2001). Interestingly, mms22A is synthetically lethal with rad52A (Araki et al., 2003), suggesting that Mms22p and Rad52p may act i n parallel pathways with overlapping functions. To assess the level o f spontaneous D N A damage and monitor the dynamics o f Rad52p-dependent D S B repair i n mms22A mutants, the percentage o f mms22A and WT cells with Rad52p foci i n the absence and presence o f a single D S B induced by H O or I-Scel, or i n 0.1% M M S was recorded (Figure 4.4D). To make sure that Rad52p foci were observed with high confidence, another marker was observed in parallel. Lisby observed that 94% o f Rad52p foci colocalize with the D S B site (Lisby et al., 2003). A n example o f Rad52p foci and D S B site colocalization is shown i n Figure 4.4C. 25-50% o f budded JfT cells, but only 5-10% o f budded mms22A cells, exhibited Rad52p foci in the presence o f D N A damage. In general, the frequencies obtained were lower than those reported by Lisby et al., who observed that 22% budded WT cells form spontaneous Rad52p foci, and 62% budded WT cells form foci after l h r exposure to 0.1% M M S (Lisby et al., 2003). This could be attributed to technical variation (e.g. subjective definition o f a Rad52p focus). In WT, the proportion o f cells with Rad52p foci increases in the presence o f D N A damage. In mms22A, the proportion o f cells containing Rad52p foci does not differ i n the absence and presence o f D N A damage. This result is different from many C I N mutants, i n which the percentage o f cells with spontaneous Rad52p foci increases, as inpol2-100, meclA (Lisby et al., 2001), top3A (71%), sgslA (41%) (Shor et a l , 2005), nupl33A (30%) and rad27A (32%) (Loeillet et al., 2005). O n the other hand, in rfalA,  Rad52p foci do not form efficiently, revealing the hierarchy in the repair  process (Lisby et al., 2004). This result suggests that Mms22p may act early i n the repair process, and may be required for the formation o f Rad52p foci.  115  In order to determine whether the lower survival rate o f mms22A i n D S B is related to slower repair kinetics, the presence o f a D S B and the completion o f repair in mms22A were monitored by southern blot and P C R analyses as i n ( A y l o n et al., 2003) (personal communication with Martin Kupiec; Figure 4.5). While the D S B is resolved by 4 hours in WT cells, the D S B persisted up to 7 hours i n mms22A cells. The level o f gene conversion was also lower in mms22A cells (60%) compared to WT cells (90%). Interestingly, the survival level as observed by colony formation was lower than the repair rate based on P C R . This discrepancy implies that some cells repair the D S B , but are still unable to survive. A similar phenomenon has been seen i n mutants compromised for checkpoint functions (e.g. rad24A, meclA) ( A y l o n et al., 2003). Although mms22A mutants have been shown to be competent for Rad53p activation (Araki et al., 2003), it w i l l be o f interest to elucidate whether mms22A mutants are defective in some aspect o f checkpoint function.  4.3.4 Mms22p interacts with replication initiation and DNA repair proteins that may constitute a novel repair pathway Mass spectrometry ( M S ) analysis o f overexpressed tagged M m s 2 2 p immunoprecipitates provided preliminary evidence that Mms22p physically interacts with Rttl01p/Cul8p and Rttl07p/Esc4p (Ho et a l , 2002). To confirm these interactions, I performed reciprocal co-immunoprecipitation (co-IP) o f endogenously expressed tagged proteins (Figure 4 . 6 A & B ) .  4.3.4.1 Mass spectrometry analysis To systematically identify additional protein interactors o f this potential novel complex, immunoprecipitation followed by M S was performed using endogenously expressed Mms22p, R t t l O l p and Rttl07p. M S analysis for M m s 2 2 p immunoprecipitates did not yield any putative interaction partners, including Mms22p itself, possibly because Mms22p is expressed at a low level (data not shown). M S o f R t t l 0 7 p immunoprecipitates identified only R t t l 0 7 p itself but no other protein. Interestingly, M S analysis o f R t t l O l p  116  immunoprecipitates identified M m s l p , a protein proposed to function upstream o f Mms22p (Figure 4.7A). To verify this interaction, reciprocal co-IP was performed using lysates extracted from logarithmically growing cells that contain no tag, M m s l p - M Y C only, R t t l O l p - H A only, and both M m s l p - M Y C and R t t l O l p - H A (Figure 4.7B). In the anti-HA IP, M m s l p - M Y C is only detected when both M m s l p - M Y C and R t t l O l p - H A are expressed. Reciprocally, in the a n t i - M Y C EP, R t t l O l p - H A is only detected in strains with M m s l p - M Y C and R t t l O l p - H A .  4.3.4.2 Y e a s t - t w o - h y b r i d a n a l y s i s  Given the common occurrence o f false-positives and false-negatives i n genomewide assays and screens, combining results using various methods often yield more informative results. Therefore, yeast-two-hybrid ( Y 2 H ) screening was performed using Mms22p, R t t l O l p and Rttl07p as baits. The Y 2 H study using Mms22p as bait identified R t t l O l p , M m s l p , M e m 1 Op, Ctf4p and several other proteins as interacting proteins (Figure 4 . 8 A & D ) . To confirm the physical interactions between M m s 2 2 p and M m s l p , reciprocal co-IP experiments were performed (Figure 4.9). Endogenously expressed M m s 2 2 p - M Y C co-immunoprecipitated with endogenously expressed M m s l - H A i n the H A - I P only when both tagged proteins were expressed. Reciprocally, endogenously expressed M m s l - H A co-immunoprecipitated with endogenously expressed Mms22pM Y C i n the M Y C - L P (Figure 4.9B). However, there was some background immunoprecipitation o f M m s l p - H A i n the H A - I P i n the absence o f M m s 2 2 p - M Y C . Therefore, reciprocal tagging was tried to avoid the background. H A - M m s 2 2 p expressed from the G A L 1 promoter was used instead. Endogenously expressed M m s l p - M Y C coimmunoprecipitated with overexpressed H A - M m s 2 2 p i n the H A - L P (Figure 4.9A): However, in the reciprocal M Y C - I P , H A - M m s 2 2 p did not co-immunoprecipitate with M m s l p - M Y C . It is possible that the overexpression o f Mms22p disrupts the localization of proteins required for its interaction with M m s l p , or it may change the stoichiometry o f its physiological protein-protein interactions. Taken together, these results strongly suggest that these two proteins not only interact genetically as reported by A r a k i et al.  117  (2003), but also physically, and likely together with R t t l O l p . It w i l l be o f interest to investigate whether the pairwise interactions among these 3 proteins are dependent on the third protein. The Y 2 H interaction o f M c m l O p , a replication initiation protein, with Mms22p is also intriguing, since mcmlO-1 and mms22A are synthetically lethal (Araki et al., 2003). Ctf4p, a Y 2 H interactor o f Mms22p, also functions in replication and cohesion, and is important for chromosome transmission fidelity (Mayer et al., 2004; Petronczki et al., 2004; Warren et al., 2004b). In addition, high-throughput Y 2 H studies showed that Mms22p (as prey) interacts with P s f l p and Psf2p, 2 o f the 4 subunits o f the G I N S complex, which is required for D N A replication initiation and progression o f D N A replication forks (Gambus et al., 2006; Hazbun et al., 2003; Takayama et al., 2003). I have been unable to confirm the physical interactions o f Mms22p with these replication proteins by co-immunoprecipitation o f endogenously tagged proteins i n logarithmic growth condition (data not shown). It is possible that these interactions represent falsepositives identified i n Y 2 H screens and do not occur i n physiological conditions, but it is also possible that the interactions are transient, occurring only at specific cell cycle stages, or only i n a very small fraction o f the total protein pool. However, because the Y 2 H interactors o f Mms22p are enriched for replication proteins (p-value = 1.84E-5, by G O Term Finder on S G D , http://db.yeastgenome.org/cgi-bin/GO/goTermFinder), and these replication proteins are not seen as common interactors with many other proteins (false-positives), these interactions may be real and functional. The Y 2 H results are also in agreement with the observation that mms22A mutants are sensitive to D N A damaging agents that cause replication-dependent D S B s , such as C P T .  4.3.4.3 G e n e t i c i n t e r a c t i o n a n a l y s i s  Genetically, mms22A interacts with mutations i n replication initiation, H R and post-replication repair genes (Pan et al., 2006; Tong et al., 2004), suggesting it may have overlapping functions i n these pathways. Unexpectedly, genome-wide S L screens using kinetochore mutants as queries revealed that mms22A also genetically interacts with  118  spc24-9 and spc24-10, temperature sensitive alleles o f a gene encoding a central kinetochore protein, by lowering their permissive temperatures (Figure 4.1 OA). A t semipermissive temperatures, both spc24-9 and spc24-10 mutants have elongated spindles and unequal distribution o f chromosomal D N A . spc24-9 is also sensitive to H U (personal communication with V i v i e n Measday). It is unlikely that Mms22p functions at the kinetochore, but the combined defects in mms22A and kinetochore mutants may sensitize cells to chromosome missegregation. Pan et al. reported that MMS22, MMS1, RTT101 and RTT107 forms a functional module or minipathway based on high congruence i n genome-wide synthetic fitness/lethal ( S F L ) interaction profiles together with the H R and iL4Z)6~-dependent repair pathways (Pan et al., 2006). Mutations i n any o f the 4 genes cause similar sensitivity to DNA-damaging treatments, and do not exhibit S F L interaction with one another, except that Pan et al. observed a synthetic fitness defect i n the rttlOlA  rttl07A mutants (Pan et  al., 2006). I did not observe a synthetic fitness defect i n the rttlOlA  rttl07A mutants in  unperturbed condition, but did observe synergistic sensitivity to M M S and H U (Figure 4.1 OB). This study and others large scale physical and genetic interaction studies (Ho et al., 2002; Pan et al., 2006; Tong et al., 2004) have generated enomorous amount o f interaction data for MMS22, MMS1, RTT101 and RTT107, which are invaluable to understanding the biological pathways o f these genes. These interactions are summarized in a network diagram (Figure 4.11).  4.3.5 R t t l O l p r e g u l a t e s M m s 2 2 p  M i c h e l et al. (Michel et al., 2003) showed that R t t l O l p has sequence homology with cullins and contains in vitro ubiquitin ligase activity, but there is as yet no known in vivo substrate. Based on the physical interactions between Mms22p and M m s l p with R t t l O l p (Figure 4.6 and 4.7), I hypothesized that Mms22p and M m s l p could be substrates o f the R t t l O l p E3 ubiquitin ligase complex. Therefore, I analyzed the steady state protein level o f M m s l p and Mms22p i n rttlOlA  119  mutants. Expression o f M m s l p is  not affected by rttlOlA (data not shown). O n the contrary, Mms22p is expressed at a much higher level in rttlOlA mutants when compared to WT (Figure 4.12A). This is consistent with the hypothesis that Mms22p is a substrate o f R t t l O l p , whereas M m s l p could regulate R t t l O l p activity. Indeed, the Mms22p expression level is similar i n rttlOlA and rttlOlA mmslA (Figure 4.12A). It w i l l be o f interest to also examine whether Mms22p expression level is affected in mmslA. To further investigate whether R t t l O l p regulates the expression level o f Mms22p through its ubiquitin ligase activity, I attempted to analyze Mms22p expression level in an rttlOl mutant that affects its ubiquitin ligase activity. R t t l O l p , like other cullins, is modified by R u b l p at a conserved lysine K791. However, K 7 9 1 is also the site o f R u b l independent modifications (Michel et al., 2003). The K 7 9 1 A mutation o f R t t l O l p was observed to reduce its in vitro ubiquitin ligase activity by 50% (Michel et al., 2003); however, another study reported that the K 7 9 1 R mutation can still complement for R t t l O l p function in a transposition assay, showing that the modification at K791 does not completely disrupt R t t l O l p function (Laplaza et al., 2004). I compared the expression level o f Mms22p i n an rttlOlA mutant containing a 2JJ, plasmid expressing either w i l d type RTT101 or rttl01-K791R under control o f the G a l promoter. In both cases, the expression level o f Mms22p was intermediate, between that observed in WT and  rttlOlA,  suggesting that both constructs partially complement the lack o f R t t l O l p (Figure 4.12A). Due to the ambiguity regarding the function o f the K791 modification, it is still difficult to conclude with certainty that M m s 2 2 p ' s expression level is affected b y R t t l O l ' s ubiquitin ligase activity. To address this ambiguity, it w i l l be useful to asses the effect o f a different mutant o f R t t l O l p . The interaction between R t t l O l p and R o c l p is essential for its ubiquitin ligase activity, since deleting the conserved Roclp-interacting domain in R t t l O l p results in complete loss of in vitro ubiquitin ligase activity ( M i c h e l et al., 2003). Therefore, comparing the expression level o f Mms22p i n an rttlOl A mutant containing a plasmid expressing either wild-type RTT101 or an rttlOl mutant lacking the R o c l p interacting domain (rttlOl-ARocT) would delineate whether the ubiquitin ligase activity of RTT101 is required for regulating Mms22p.  120  While the above experiments looked at the steady state expression level o f Mms22p in logarithmic growth, it was o f interest to examine the kinetics o f Mms22p degradation i n the presence or absence o f R t t l O l p . Mms22p was expressed from the galactose promoter i n medium containing galactose, and the expression was then shut off by growth i n glucose medium. The level o f Mms22p was monitored at 20 m i n intervals for 100 m i n (Figure 4.12B). Interestingly, Mms22p levels increased to a higher level during the induction period in rttlOl A mutants. However, the degradation rate o f Mms22p was not reduced in rttlOl A. It is possible that Mms22p is also degraded by an RttlOlp-independent pathway. In this experimental condition, however, the Mms22p could still be translated from residual m R N A transcripts after promoter shut off. Therefore, i n future experiments, cycloheximide, a drug that inhibits protein translation, should be added when the culture is released into glucose. R t t l O l p could regulate the degradation o f Mms22p i n a cell-cycle dependent manner or in response to D N A damage. I therefore analyzed whether Mms22p and R t t l O l p are induced or modified under various conditions. Microarray analysis revealed that MMS22 m R N A expression is induced 5 minutes after 0.02% M M S addition and 20 minutes after heat shock (Gasch et al., 2001). Western blot analysis o f Mms22p i n different cell cycle stages or 0.01% M M S for 15 m i n at different cell cycle stages showed similar Mms22p expression level (data not shown). However, R t t l O l p showed a slower-migrating band in the presence o f H U and nocodazole (Figure 4.13), suggesting it may be modified in a cell cycle-specific manner. Further experiments are required to distinguish whether these modifications are cell cycle specific, or whether they are side effects related to drug treatment.  121  4.4 Discussion B y integrating phenotypic, genetic and physical interaction data from the literature and from this study, I confirmed that mms22A is defective i n cell cycle progression and D N A D S B repair. Co-immunoprecipitation experiments indicate that Mms22p, R t t l O l p and M m s l p physically interact with each other. These data support that Mms22p functions with M m s l p and R t t l O l p in an E3 ubiquitin ligase. Indeed, the expression o f Mms22p is regulated by R t t l O l p , and it is possible that Mms22p is a substrate o f the R t t l O l E3 ligase. Since M m s l p expression level is not affected by R t t l O l p , M m s l p may serve as a specificity factor the E3 ubiquitin ligase (see below). This work leads to the proposal o f a model in which R t t l O l p may regulate Mms22p and other protein levels i n response to D N A damage.  4.4.1 Conservation of the RttlOlp complex? While there is as yet no confirmed substrate for the R t t l O l p ubiquitin ligase, clues regarding its function may be gained from knowledge about other cullins. C u l l i n serves a scaffolding function: it interacts through its N-terminal domain with a substrate specificity factor, and through its conserved globular C-terminal domain (called cullin homology domain) with the R I N G finger protein to form the catalytic core. In addition to C u l l p / C d c 5 3 p / C u l A p i n S C F , budding yeast has 2 additional cullins: Cul3p/CulBp and Rttl01p/Cul8p/CulCp, whereas humans have 4 additional cullins: C U L 2 , C U L 3 , C U L 4 A , C U L 4 B , and C U L 5 . R t t l O l p displays protein sequence similarity to all o f the human cullins. However, it is unknown whether R t t l O l p is the functional ortholog to any of the known human cullins. A r a k i et al. (Araki et al., 2003) claimed that M m s l p has weak similarity to R a d l 7 p and D d c l p . Recently, M m s l p was found to have homology to human D D B 1 , the adaptor o f C U L 4 A (Mathias Peter, personal communication). The damaged-DNA binding proteins, D D B 1 and D D B 2 , recognize damaged D N A and are important for global genome repair ( G G R ) , one pathway i n N E R that repairs the D N A damage across the entire genome. X P patients (in the X P E complementation group) have mutations in  122  DDB2. D D B 2 , through its binding to D D B 1 , interacts with C U L 4 A and R O C 1 . In response to U V , C U L 4 A is post-translationally modified, which stimulates the ubiquitin ligase activity o f the D D B complex to ubiquitylates X P C (Matsuda et al., 2005). Such ubiquitin modification has non-proteolytic function, but instead signals the cell to a specific D N A repair pathway. Genetic analysis suggests that the R t t l O l p complex functions downstream o f P C N A and controls the function o f the translesion D N A synthesis (TLS)-polymerase zeta, allowing the replication bypass o f damaged templates during D N A replication. Mms22p exhibits weak homology to S. pombe T a z l , a telomere-binding protein that is required for efficient replication fork progression through the telomere (Miller et al., 2006). tazl A mutants have stalled replication forks at telomeres and telomere sequences placed internally on a chromosome. T a z l may recruit helicases to facilitate unwinding o f the G-rich telomere repeats. T a z l is required to protect telomeres from NHEJ-mediated telomere fusions, and to prevent chromosomal entanglements and missegregation at cold temperatures (Miller et al., 2005). Human T R F 1 and T R F 2 are putative orthologues o f T a z l , and may also orchestrate fork passage through human telomeres. However, no orthologue o f T a z l has been identified i n S.  cerevisiae.  It would be o f interest to investigate i f Mms22p has a role to facilitate replication fork progression through G-rich regions or other barriers. Interestingly, R t t l O l p and Rttl07p also have a role i n facilitating replication fork restart through alkylated and r D N A regions, respectively (Chin et al., 2006; Luke et al., 2006). O n the other hand, A r a k i et al. (Araki et al., 2003) found that Mms22p has weak similarity to Rad50p, a component o f the M R X complex. Mutants o f the M R X complex and M M S 2 2 display similar drug sensitivity profiles, but further studies are required to determine whether Mms22p functions in a similar way as Rad50p.  123  4 . 4 . 2 Dia2p may play a redundant role with the RttlOlp complex in replication regulation L i k e rttlOl A, dial A mutants accumulate i n S / G 2 / M , exhibit constitutive activation o f Rad53p, increased foci o f D N A repair proteins, elevated G C R and C L N as found i n our screens (Chapter 2), and are unable to overcome M M S - i n d u c e d replicative stress. Dia2p, a F-box protein i n the S C F , is required for stable passage o f replication forks through regions o f damaged D N A and natural fragile regions, particularly the replication fork barrier (RFB) o f r D N A repeat loci (Blake et al., 2006). The synthetic lethal interaction profile o f dia2A mutants clusters with mutants i n D N A replication and repair (rad51A, rad52A, rad54A, rad57A, hpr5Alsrs2A),  the replication checkpoint  (csm3A, toflA, mrclA), the alternative R F C (dcclA, ctf8A, ctfl8A), the M R X complex, post-replicative repair (rad5A, rad!8A), and rttlOl A, rttlOl A and mmslA (Blake et al., 2006). S C F  D , a 2  may modify or degrade protein substrates that would otherwise impede  the replication fork i n problematic regions o f the genome. Interestingly, Dia2p binds to replication origins after origin firing, possibly to reset them for use i n the next S-phase (Koepp et al., 2006). It is possible that Dia2p acts i n a redundant fashion with R t t l O l p ubiquitin ligase to modify or eliminate substrates at the replication fork.  4 . 4 . 3 Identifying targets for RttlOlp ubiquitin ligase Although R t t l O l p has in vitro ubiquitin ligase activity, and interacts with the R I N G finger protein R o c l p and the E 2 , Cdc34p, no in vivo substrate has been confirmed. The next important goal i n characterizing the function o f the R t t l O l p complex is to identify its target substrates. Ubiquitin modification o f targets b y the R t t l O l p complex may lead to cell-cycle or D N A damage specific proteolysis, or may determine the D N A repair pathway used b y the cell (reviewed i n (Huang and D'Andrea, 2006)). This study suggests that Mms22p is a component o f the E3 complex, but it could also be a substrate. Autocatalytic degradation has been described for other ubiquitin ligases. For instance, the B R C A l - B A R D I complex can autopolyubiquitylate i n response to D N A damage, and this autoubiquitylation stimulates its E3 ligase activity to ubiquitylate histone proteins (Huang  124  and D'Andrea, 2006). In addition, D D B 2 is also ubiquitylated b y the D D B - C U L 4 A complex i n response to U V (Matsuda et al., 2005). Physical interactors with Mms22p identified from genome-wide methods, i n particular the replication proteins such as M c m l O p , Ctf4p, P s f l p and Psf2p, are candidate substrates o f the R t t l O l p ubiquitin ligase. The G I N S complex, including P s f l p and Psf2p, is required for D N A replication initiation and progression o f D N A replication forks (Gambus et al., 2006; Hazbun et al., 2003; Takayama et al., 2003). The G I N S complex allows the M C M complex to interact with the replisome progression complexes (RPCs), which include Ctf4p, among other replication proteins. Interestingly, R P C s also interact with M c m l O p (Gambus et al., 2006). Since mms22A mutants exhibit aneuploidy and some cells accumulate >2N D N A contents, it is possible that Mms22p is involved in the proteolysis o f some replication proteins help to ensure that D N A is not re-replicated. It is known that budding yeast employ multiple regulatory mechanisms, including proteolysis o f important factors, to serve this function. The replication licensing factor Cdc6p is known to be degraded through S C F  C d c 4  in S phase. In addition, Orc2p and  Orc6p, components o f the origin recognition complex, are phosphorylated by S phase c y c l i n / C D K to inhibit pre-RC reassembly. Furthermore, another replication licensing factor C d t l and the M C M complex are exported from the nucleus (Guardavaccaro and Pagano, 2004; Pintard et al., 2004). Similarly, human M c m l O p is phosphorylated and degraded in a cell cycle-dependent manner (Izumi et al., 2001). In human cells, overexpression o f C d t l leads to re-replication and polyploidy, and have been described in many cancers (Feng and Kipreos, 2003). It w i l l be o f interest to investigate whether the R t t l O l p complex and their targets are involved i n such function. Recently, both Mms22p and R t t l O l p were found to physically interact with H 3 (Hhtlp) and H 4 (Hhflp) (Krogan et al., 2006) (Figure 4.11). In addition, M m s 2 2 p interacts with H 2 B (Htb2p), while R t t l O l p interacts with H 2 A (Hta2p) (Krogan et al., 2006), suggesting the core histones may be potential substrates. Dephosphorylation o f H 2 A is necessary for efficient removal o f the cell cycle checkpoint (Keogh et al., 2006), but H 2 A may also be regulated by degradation upon completion o f D N A repair.  125  In addition to the candidate approach, unbiased target screening methods that have been described for substrate specificity factors should also be applicable for cullins, though the number o f substrates for cullins may be greater than that for specificity factors. A preliminary screen was performed using a system described by Deanna Koepp (personal communication), in which an ADE3-gene. fusion plasmid library was transformed in a strain lacking the gene o f interest i n the ubiquitin machinery (e.g. RTT101). The color o f the yeast cells depends on the stability o f the fusion protein. A t the same time, a plasmid containing RTT101 was transformed, but with no selection. This leads to loss o f the RTT101 plasmid i n some cells during colony formation. The generation o f sectored colonies indicates that the stability o f the fusion protein is affected by the presence or absence o f R t t l O l p . Similarly, i n a microscopic screening system described by D a v i d Toczyski (personal communication), a GFP-fusion protein signal was compared between WT and strains lacking the gene o f interest. This method has successfully identified and confirmed substrates for the F-box protein G r r l p (David Toczyski, unpublished). Since the cullins i n yeast do not seem to be functionally redundant based on their differences in phenotypes, substrate screening for R t t l O l p w i l l shed insight to its biological functions.  126  T a b l e 4.1 T y p e s D N A lesions generated b y v a r i o u s D N A d a m a g i n g agents DNA damaging agent DNA lesion(s)  Methyl methanesulfonate (MMS) Hydroxyurea (HU) Camptothecin (CPT)  Ultra-violet (UV) radiation 4-nitroquinoline-l-oxide (4NQO) Ionizing radiation (IR)  produces predominately 7-mehtylguanine and 3-methyladenine, which block DNA replication; as well as a small percentage of 06methylguanine and 04-methylthymine, both of which cause base mispairing a ribonucleotide reductase inhibitor, inhibits DNA replication by depleting dNTPs traps topoisomerase I (Topi) in the cleavage complex, causing singlestranded DNA (ssDNA) nicks that inhibit DNA replication and can be converted into double strand breaks (DSBs) by the advancing replication fork induces primarily cyclobutane pyrimidine dimers and photoproducts, which are efficiently targeted by the nucleotide excision repair (NER) pathway a UV mimetic agent, introduces bulky DNA adducts that are also mainly removed by NER induces DSBs that are replication-independent  127  T a b l e 4.2 1 l^ist of yeast strains used in Chapter 4 Strain Genotype YKY90 MATa/MATa ura3-52/ura3-52 trplA-63/trplA-63 his3A-200/his3A-200  YKY570  YKY332  YPH499 YKY62 YKY64 YKY104 YKY108 YKY210 YKY248 YKY249 YKY253 YKY256 YKY260 YKY264 YKY269 YKY807  YTK1364  YKY754 /MK203 YKY755 YKY848 YKY713  Reference This study  leu2A-l/leu2A-I ade2-10l/ade2-Wl lys2-801/lys2-801 CFIII(CEN3.L)URA3 SUP 11 mms22A::HIS3/ mms22A::HIS3 This study MATa/MATa ura3-52/ura3-52 trplA-63/trplA-63 his3A-200/his3A-200 leu2A-l/leu2A-l ade2-l01/ade2-101 lys2-801/lys2-801 CFIII(CEN3.L)URA3 SUPU rttWlAr.TRPU rttlOlAr.TRPl This study MATa/MATa ura3-52/ura3-52 trplA-63/trplA-63 his3A-200/his3A-200 leu2A-l/leu2A-l ade2-101/ ade2-101 lys2-801/ lys2-801 CFIII(CEN3.L)URA3 SUP11 rttl07A::TRPl/rttl07A::TRPl Hieter lab MATa ura3-52 trplA-63 his3A-200 leu2A-J ade2-10J lys2-801 This study MATaura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 CFII1(CEN3.L)-URA3 SUPU mms22A::HIS3 MATa ura3-52 trplA-63 his3A-200 leu2A-J ade2-101 lys2-801 This study mms22A::HIS3 This study MATaura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 CFIII(CEN3.L)-URA3 SUPU mms22A::HlS3 mad2A::HIS3 This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 CFIII(CEN3.L)-URA3 SUPU mms22A::HIS3 rad9A::LEU2 This study MATa ura3 trplA-63 his3 leu2A-l mms22A::HIS3 mec3A::kanMX This study MATa ura3 trplA-63 his3 leu2A-l mms22A::HIS3 mrclAr.kanMX MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-l0l Iys2-80J pRS414-TRPlThis study This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 Iys2-80J rad52A::LEU2 pRS414-TRPl This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 mms22A::HIS3 pRS4l4-TRPl This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-10l lys2-801 pJH132(pGAL-HO)-TRPl This study MATa ura3-52 trplA-63 his3A-200 leu2A-J ade2-l01 lys2-801 rad52A::LEU2pJH132(pGAL-HO)-TRPl This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 mms22A::HIS3pJH132(pGAL-HO)-TRPl MATa ade2-l barl::LEU2 trpl-l LYS2 RAD5 RAD52-CFP ura3::3xURA3- (Lisby et tetOxll21-Sce(ura3-1) his3-U,15::YFP-LacI-his3-x leu2-3,l 12::LacOal., 2003) LEU2HO-iYCL018W0eu2-3,l 12) TetR-RFP(iYGLl 19W) pJH1320(pGALlSceI)-ADE2 URA3 MATa ade2-l barl.:LEU2 trpl-l LYS2 RAD5 RAD52-CFP ura3::3xURA3- This study tetOxll2 I-Sce{ura3-1) his3-ll,15::YFP-LacI-his3-x leu2-3,l 12::LacOLEU2HO-iYCL018W0eu2-3,U2) TetR-RFP(iYGLl 19W) mms22A::kanMX pJH1320(pGAL-lSceI)-ADE2 URA3 (Aylon et MATa-inc ade2 ade3::GALHO ura3::HOcs leu2-3,112 his3-ll,13 trpl-l lys2::ura3::HOcs-inc(RB) al., 2003) MATa-inc ade2 ade3::GALHO ura3::H0cs leu2-3,112 his3-11,13 trpl-l (Aylon et al., 2003) lys2:: ura3: :HOcs-inc(RB) rad52A: :LEU2 This study MATa-inc ade2 ade3::GALHO ura3::H0cs Ieu2-3,U2 his3-ll,13 trpl-l lys2::ura3::HOcs-inc(RB) mms22A::kanMX MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 MMS22- This study 13MYC::HIS3 RTT101-3HA::TRP1  128  YKY435 YKY721 YKY461 YKY413 YKY447 YKY690 YTK1168 YTK1132 YKY527 YTK1140 YTK1345 YTK1375 YKY820 YKY821 YKY822 YKY824 YKY831 YKY836 YKY297 YKY325 YKY657 YKY642 YKY648 YKY767 YKY956  MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 RTT101- This study 3HA::TRP1 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 MMS22- This study 13MYC:HIS3 RTT107-3HA::TRP1 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 RTT107- This study 3HA::TRP1 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 RTT101- This study 13MYC:: TRP1 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 RTT107- This study 13MYC:: TRP1 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-80l MMS22- This study 13MYC:: HIS3 This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-Wl lys2-801 MMS13HA::kanMXRTT101-13MYC:: TRP] This study MATaura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 MMS13HA::kanMX MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 HIS3-pGAL-This study 3HA-MMS22 This study MATaura3-52 trplA-63 his3A-200 leu2A-l ade2-10l lys2-801 MMS113MYC::kanMX This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 MMS113MYC::kanMX HIS3-pGAL-3HA-MMS22 This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 MMS13HA::kanMXMMS22-13MYC::HIS3 V. MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-80l spc248::kanMX Measday V. MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-Wl Iys2-80J spc249::kanMX Measday V. MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-10l lys2-801 spc24Wr.kanMX Measday This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-10l lys2-801 spc248: .kanMX mms22A::HIS3 This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 spc249::kanMX mms22A::HIS3 This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-l0l lys2-801 spc24Wr.kanMXmms22A::HIS3 This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 rttl01A::TRPl This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 rttW7A::TRPl This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 rttlOl A: :TRP1 mms22A::HIS3 This study MATa ura3-S2 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 rttl07A::TRPl mms22A::HIS3 This study MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 rttl07A::TRPl rttlOl A:: kanMX MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 MMS22- This study 13MYC::HIS3 rttlOl A:: TRP 1 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 MMS22- This study  129  YKY956 YKY782  13MYC::HIS3 rttlOlAr.TRPl pYES-RTT101-URA3 MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 lys2-801 MMS22- This study 13MYC::HIS3 rttlOlA::TRP1 pYES-rttl01-K791R-URA3 • MATa ura3-52 trplA-63 his3A-200 leu2A-l ade2-101 Iys2-80J HIS3-pGAL-This study 3HA-MMS22 rtt!01A::TRPl  130  T a b l e 4.3 Q u a n t i f i c a t i o n o f c h r o m o s o m e loss ( C L ) , n o n - d i s j u n c t i o n ( N D J ) a n d c h r o m o s o m e g a i n ( C G ) b y half-sectored assay Total no. C L freq. IS ».l lrc(|. No. pink-red No. whiteStrain colonies (fold over (fold over colonies red colonies counted WT) WT) 8.7E-5 8.7E-5 (Shero et WT diploid N/A N/A (Shero et N/A al., 1991) al., 1991) mms22A 3.0E-3 3.4E-4 11890 36 4 (35X) (4X) mms22A rttlOlA rttlOl A rttl07A rttl07A  14600  40  2.7E-3 (3IX)  32  17730  40  2.7E-3 (26X)  i6  131  ;  1  No. pinkwhite colonies N/A  CGfreq.  N/A '.  3  2.5E-4  2.2E-3 (25X)  93  6.4E-3  9.0E-4 (10X)  52  2.9E-3  DNA damage and repair mechanisms (reprinted from (Hoeijmakers, 2001) Genome maintenance mechanisms for preventing cancer, Nature, 411, 366-374, Copyright 2001, with permission from Macmillan Publishers Ltd) F i g u r e 4.1  Common DNA damaging agents (top); examples of DNA lesions induced by these agents (middle); and most relevant DNA repair mechanism responsible for the removal of the lesions (bottom). (6-4)PP and CPT, 6-4 photoproduct and cyclobutane pyrimidine dimer, respectively (both induced by UV light); EJ, end joining. lamaqinq agent  X-ray* Oxygen radicals AlKylaung  agent*  Spontaneous reactions  t Xu ro au yrsaycnsl Rcpbc an U V k g m o in A M i t m Po t y c y d c i a r o m a c t i e r o r s o l s P t , M M C ) hydo ioab ions  Urasctile i Bu m iDo eu s ftb ra n d cros se n ilak Jk 6 -4JPP e l s t r a n d t x k l y a d d u c t 8 O x o g u a n n i c C P D Sn ige l strand ore-atl B ap aa etr-iexcIB sio£ nR)Nurc e lp oa d tirie-e|N x£ csia inRJR ep co irN ja» tR.naE lJ) re e re am ri n Abasit  132  T-CInM srim ac th s e o t i n Dee lo tin t M simac th repari A-G Mbmatch  F i g u r e 4.2 T w o - d i m e n s i o n a l h i e r a r c h i c a l c l u s t e r i n g o f drugs ( h o r i z o n t a l ) a n d yeast d e l e t i o n mutants (vertical) that s h o w s e n s i t i v i t y to at least 1 d r u g b a s e d o n g e n o m e - w i d e d r u g s e n s i t i v i t y screens, a n d o v e r l a i d w i t h the C I N screen results. T h e c l u s t e r i n g w a s p e r f o r m e d u s i n g the p r o g r a m C l u s t e r 3.0 a n d d i s p l a y e d i n J a v a T r e e V i e w ( v e r s i o n 1.0.8) ( E i s e n e t a l . , 1998).  A. An overview of all deletion mutants and drugs. The severity of drug sensitivity is indicated by color, with bright red indicating high sensitivity, darker red showing milder sensitivity, and black representing the same sensitivity as wi