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Identification of novel factors required for chromosome segregation in budding yeast Cheng, Benjamin 2005

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IDENTIFICATION  OF N O V E L FACTORS  REQUIRED  F O R C H R O M O S O M E S E G R E G A T I O N IN B U D D I N G YEAST  BY BENJAMIN CHENG B . S c , Simon Fraser University, 1998  A Thesis Submitted i n Partial Fulfillment o f the Requirements for the Degree o f D O C T O R OF PHILOSOPHY in The Faculty o f Graduate Studies (Medical Genetics)  The University o f British Columbia October 2005  © Benjamin Cheng, 2005  Abstract During the course o f the mitotic cell cycle, the genetic material must be faithfully replicated and segregated to daughter cells. After D N A replication when chromosomes have been duplicated, each pair o f identical sister chromatids must remain tethered together until all pairs o f sister centromeres have attached to the mitotic spindle i n a b i oriented manner, a state termed metaphase. Once metaphase has been successfully achieved, the initiation o f anaphase can take place, and sister chromatids are pulled apart to the two daughter cells. Errors i n this process lead to chromosome missegregation (chromosome loss or non-disjunction) and result i n aneuploidy, which may have deleterious effects. Processes important in chromosome segregation fidelity include kinetochore attachment to the spindle and sister chromatid cohesion. A genome wide two hybrid screen using SGT1 as the "bait" identified a previously uncharacterized open reading frame, Y D R 0 1 4 W (RAD61) that, when deleted, missegregated a chromosome fragment. Y D R 0 1 4 W corresponded to the gene encoding the complementation group, CTF6, and was also recently characterized as RAD61 i n a screen for deletion mutants sensitive to ionizing radiation. rad61A diploid mutant strains displayed a G 2 / M progression delay dependent on Mad2p and were hypersensitive to D N A damaging agents. Rad61p localizes to the nucleus and a fraction binds chromatin. Rad61p is not a core component o f the yeast kinetochore and is not required for homologous recombination repair o f D N A damage, but is important for sister chromatid cohesion. U s i n g co-immunoprecipitation and mass spectrometry analysis, we identified a protein-protein interaction between Rad61p and D e d l p , an R N A helicase o f the D E A D  11  box family that has important roles i n initiation o f translation and m R N A splicing. D e d l p binds chromatin and may have direct roles i n chromosome biology. A temperature sensitive allele o f the essential DED1 gene causes an increased rate o f chromosome missegregation. rad61A and dedl temperature sensitive alleles displayed conditional synthetic lethality, indicating that the interaction is functionally significant within yeast cells. Taken together, these results suggests that Rad61p and D e d l p function together i n the nucleus for processes that are important for sister chromatid cohesion and for chromosome segregation.  iii  T A B L E OF CONTENTS Abstract...  ii  Table o f Contents  iv  List o f Tables  viii  L i s t o f Figures  ix  List o f Abbreviations  xi  Acknowledgements  xiv  CHAPTER 1. INTRODUCTION: CHROMOSOME SEGREGATION IN YEAST AND HUMANS... 1 Introduction  2  Consequences o f Aneuploidy i n Humans  2  Budding yeast as a model organism  6  The C e l l Cycle and Genome Stability  10  D N A Replication and Genome Stability  14  The Mitotic Spindle and Chromosome Segregation  17  The Centromere and Kinetochore in Budding Yeast  20  Sister Chromatid Cohesion  24  Additional proteins required for sister chromatid cohesion  28  Meiotic sister chromatid cohesion  32  iv  Sister Chromatid Cohesion and D N A damage checkpoint and repair  33  Checkpoints that Monitor Events and Damage during the C e l l C y c l e . . .36 D N A Damage Checkpoint / S phase checkpoint  37  The Spindle Assembly Checkpoint  39  Chromosome Transmission Fidelity Mutants and Characterizing N o v e l Genes Involved i n Chromosome Segregation.... 41 Overview and Scope o f the Thesis  CHAPTER 2. Analysis oiSGTl genetic and physical interactions and identification of a factor involved in chromosome segregation, RAD61 / CTF6  44  46  Introduction  47  Materials and Methods  48  Results  55  Identification o f H i g h C o p y Suppressors o f sgtl-3 (Suppressors o f the G 2 allele o f SGT1, SOGs)  55  SOG2 and SOG3 do not encode S g t l p binding proteins and are not required for chromosome segregation  56  Identification o f an O R F , Y D R 0 1 4 W , as an S G T 1 two hybrid interacting gene required for chromosome transmission fidelity  60  Y D R 0 1 4 W corresponds to a ctf mutant (CTF6) as w e l l as a rad mutant (RAD61)....  64  rad61A mutants exhibit a cell cycle delay i n G 2 / M that is dependent on the spindle assembly checkpoint  65  Rad61p is a stable nuclear protein  67  A sub-fraction o f Rad61p binds chromatin  71  RAD61 is not involved in Homologous Recombination to repair D N A damage  73  Discussion  78  CHAPTER 3. Approaches to dissecting the role of Rad61p in chromosome segregation  81  Introduction  82  Materials and Methods  83  Results  90  rad61A has genetic interactions with kinetochore and cohesion gene mutations  90  Rad61p is involved i n sister chromatid cohesion and potentially i n establishment  96  B y ChlP-chip analysis Rad61p associates with regions around centromere D N A  100  Interaction between Rad61p and D e d l p  103  D e d l p binds chromatin and has a role in genome integrity  106  Discussion  113  CHAPTER 4. Conclusions and Discussion  121  Approaches to Identifying novel components involved in chromosome segregation  122  Genome W i d e Two-hybrid screen ofSGTl  identifies RAD61  122  Rad61p and its role i n sister chromatid cohesion  124  Rad61p and D e d l p interaction  125  D e d l p and its role i n chromosome segregation  126  vi  R N A Helicases in Human Cancers  127  RAD61 and potential human homologues  130  Conclusion  130  References  133  vn  LIST OF T A B L E S C H A P T E R II Table 2-1  List o f yeast strains used in this chapter  53  Table 2-2  Genome Wide two-hybrid interactions with SGT1  61  Table 2-3  RAD61 mutant alleles i n the ctf mutant collection  64  Table 2-4  Double Strand Break Induction - Rad52 foci formation is unaffected i n rad61A cells  77  Table 3-1  List o f yeast strains used in this chapter  87  Table 3-2  rad61A genetic interactions  90  Table 3-3  D i p l o i d - S L A M Results  94  Table 3-4  GST-Rad61 purified protein incubated with yeast lysate -  C H A P T E R ni  Table 3-5  peptides that were isolated in multiple copies  104  DED1 mutants and their sectoring phenotypes  109  viii  LIST OF FIGURES C H A P T E R 1: Figure 1-1  The budding yeast cell and chromosome cycle  8  Figure 1-2  The cohesin complex forms a ring like structure  25  C H A P T E R 2: Figure 2-1  Suppression o f the temperature sensitivity o f sgtl-3 by overexpression o f SOG2 and RPS29A  Figure 2-2  57  sog2 and sog3 deletion mutants show similar sensitivity to benomyl as w i l d type strains  59  Figure 2-3  Sog2p and Sog3p do not co-IP with S g t l p i n yeast extracts  60  Figure 2-4  rad61A mutants sector  62  Figure 2-5  Co-immunoprecipitation from yeast extracts does not indicate an interaction between R a d 6 1 - 1 3 M Y C p and S g t l - 6 H A p  Figure 2-6  rad61A / rad61A diploid cells have a G 2 / M accumulation dependent on Mad2p and are resistant to benomyl  Figure 2-7  Figure 2-9  66  R a d 6 1 - 1 3 M Y C p is stable through the cell cycle with a long half-life  Figure 2-8  63  69  Rad61p localizes to the nucleus b y indirect immunofluorescence and live cell imaging  71  A small fraction o f Rad61p binds chromatin  73  ix  Figure 2-10  rad61A strain is not sensitive to overexpression o f H O endonuclease and does not affect Rad52p foci formation  76  Figure 3-1  Schematic o f the d i p l o i d - S L A M technique  92  Figure 3-2  Clustering o f d - S L A M genetic interactions with  C H A P T E R 3:  genome wide S G A screens from Tong et al., 2004 Figure 3-3  rad61A strains display a cohesion defect that is likely a result o f defective establishment  Figure 3-4  99  Rad61p by ChlP-chip analysis is specifically enriched at Centromere D N A  Figure 3-5  95  102  Protein-protein interactions o f Rad61p and D e d l p and unconfirmed interactions o f Rad61p with A l t l p and V p s 3 p . . . . 105  Figure 3-6  D e d l p binds chromatin but the majority o f the protein is i n the cytoplasm  107  Figure 3-7  dedl-55 mutants sector at 35°C  110  Figure 3-8  dedl-51 strains have a cell cycle defect and potential sister chromatid cohesion defect  112  Potential Human Homologues o f RAD61  131  C H A P T E R 4: Figure 4-1  LIST O F ABBREVIATIONS APC/C-  Anaphase Promoting Complex / Cyclosome  bp-  base pairs  CEN-  centromere  CLN -  Chromosome Instability  CMP-  Chromatin Immunoprecipitation  CDK-  C y c l i n Dependent Kinase  CF-  Chromosome Fragment  CFP-  Cyan Fluorescent Protein  CKI-  C D K Inhibitor  CTF -  Chromosome Transmission Fidelity  DMSO-  Dimethylsulphoxide  DSB -  Double Strand Break  EMS-  Ethylmethanesulfonate  FACS-  Fluorescence Activated C e l l Sorting  Gl -  Gap 1 (Growth before D N A replication phase o f cell cycle)  G2-  Gap2 (Growth after D N A replication phase o f cell cycle)  GAP -  GTPase activating protein  GEF-  Guanine nucleotide Exchange Factor  GFP -  Green Fluorescent Protein  GDP -  Guanine Di-Phosphate  GTP -  Guanine Tri-Phosphate  HR -  Homologous Recombination  xi  IP-  Immunoprecipitation  LAC-  Lactose  LOH-  Loss O f Heterozygosity  M -  Mitosis  Mbp -  M e g a base pairs  MIN-  Microsatellite Instability  MMS -  Methylmethanesulfonate  MRX-  Mrellp/Rad50p/Xrs2p  NHEJ-  Non-Homologous End Joining  ORC-  Origin Replication Complex  ORF -  Open Reading Frame  PCNA-  Proliferating C e l l Nuclear Antigen  PJXK-  phosphoinositide 3-kinase related kinases  pre-RC -  pre-Replicative Complex  RFC -  Replication Factor C  RPA -  Replication Protein A  RSC-  Remodels the Structure o f Chromatin  S-  Synapsis ( D N A replication phase o f cell cycle)  SCF-  S k p l p / C u l l i n / F-box protein  SDL-  Synthetic Dosage Lethality  SLAM-  Synthetic Lethal Analysis on Microarrays  SPB-  Spindle Pole B o d y  ssDNA -  single stranded D N A  TET -  Tetracycline  UBC -  Ubiquitin Carrying Enzyme  UTR -  Untranslated Region  VFP -  "Venus" yellow fluorescent protein  YFP -  Y e l l o w Fluorescent Protein  YPD -  Yeast Proteome Database  YPD -  Yeast Extract Peptone Dextrose  xiii  Acknowledgements M a n y people were instrumental i n this thesis being completed. I would like to take some time now to acknowledge and thank them. I would like to thank m y supervisor, P h i l Hieter, for his advice, encouragement and guidance during the course o f both m y thesis work and the subsequent thesis writing. P h i l has provided many contacts that have turned into collaborations that have helped this work progress. I am appreciative o f his generosity through the years that I have been a member o f his lab. P h i l also provided an atmosphere conducive to research b y having excellent scientist that facilitated interactions and I would like to thank the members o f the Hieter laboratory, past and present. I would especially like to thank Katsumi Kitagawa, who as a post-doctoral fellow i n the lab began this work and trained me to take over the work. I learned a great deal o f yeast genetics and biochemistry from Katsumi and I am thankful o f the time he took to work with me. I would like to thank the members o f the Hieter lab o f the past especially Isabelle Pot (my birthday buddy), Andrew Page, Melanie Mayer, Kathy Hyland, Jean-Michel Flaman, Kristin Baetz, Scott Givan, Daniel Kornitzer and V i v i e n Measday. V i v i e n read a draft o f the thesis and provided valuable critical feedback. Thanks also go to members o f the present lab for valuable interactions (including asking for constant updates on the thesis writing!), thanks to Teresa K w o k , Irene Barrett, Victoria Aneliunas, Karen Y u e n , Shay Ben-Aroya, K i r k M c M a n u s , Jan Stoepel, B e n Montpetit, and Dave Thomson (Phil's assistant). I would also like to thank m y scientific colleagues that have contributed to this work. W e participated in collaborations with Gerard Cagney i n Stan Fields' lab at the University o f Washington who performed the initial two hybrid screen that identified  xiv  Rad61p, Xuewen Pan i n Jef Boeke's lab at Johns Hopkins who performed the diploidS L A M , M a r k Flory i n R u d i Aebersold's lab at the University o f Washington who performed the tandem mass spectrometry analysis, and Anthony Borneman i n M i c h a e l Snyder's lab at Y a l e University who performed the C M P - c h i p analysis. I would like to thank m y committee members, Carolyn Brown, A n n Rose, and M i c h e l Roberge, who provided guidance and advice through m y thesis work and encouraged me to finish and write. They also provided valuable feedback on m y thesis draft. I would like to acknowledge and thank all m y friends that have helped me through these years as I have been working on m y thesis. I would especially like to thank people that I have played Ultimate with i n the past including members o f Likastik, Lab Ratz, and Idle Hands, for allowing me a chance to play and have fun (and drop pulls). Thanks also go to people that I have played board games with, members o f the North West Diplomacy Group, Alfred L o h , John Lee, and many others. Without these two outlets the sometimes frustrating work o f research would have been that much more frustrating and more difficult. I would also like to thank members o f the Grandview Calvary Baptist Church, a place that I have felt blessed to be a part o f in the last two years. I would finally like to thank the people that are most important to me, m y family. I would like to thank m y parents, Moses and Dorcas Cheng, who have supported me unconditionally through the many years o f school. I would like to thank m y siblings, M a y , C a l v i n and Joshua who encouraged me through m y degree. Finally I would like to thank m y wife, Angela, who during the times that I was ready to stop and run away, encouraged me and through her patience, willed me into getting this thing done! She has  xv  been and continues to be a wonderful blessing i n m y life. I would like to dedicate this thesis to her and to our little genetics experiment growing inside her! During the course o f this work I received funding from N S E R C , C I H R and the K i l l a m Foundation and I would like to acknowledge their generous support.  xvi  C H A P T E R 1: INTRODUCTION  CHROMOSOME SEGREGATION IN YEAST AND HUMANS  1  Introduction The proper replication and segregation o f the genetic material i n dividing cells is a fundamental process i n biology. Cells have evolved mechanisms to ensure that chromosomal D N A is replicated faithfully, packaged into chromatin (consisting o f D N A , histones, and associated proteins), held together as sister chromatids, and then properly segregated, one copy to each o f the daughter cells. Errors i n this process can lead to aneuploidy, a state i n which a cell does not have the proper complement o f chromosomes. In aneuploid cells, one or more chromosomes are missing or there is an excess number o f chromosomes. Aneuploidy can cause the uncovering o f recessive mutations, or result i n cell death or adverse growth effects. Aneuploid cells and genomic instability are also important factors that contribute to human diseases such as D o w n Syndrome and cancer. Studying the factors that govern genomic stability is thus important not only for understanding basic cell processes, but also for understanding human disease and for developing potential therapeutics.  Consequences of Aneuploidy in Humans Genomic instability and aneuploidy are important factors i n tumourigenesis. The majority o f solid tumours contain aneuploid cells. Tumour cell lines have an increased rate o f aneuploidy generation and not just an increased number o f cells that are aneuploid. The vast majority o f colon tumours (>80%) are classified as having a chromosome instability (CEN) phenotype (Nowak et al. 2002). One model for how aneuploidy could contribute to tumourigenesis is that an increased rate o f aneuploidy could act to increase the rate o f loss o f heterozygosity ( L O H ) thereby increasing the  2  chances o f uncovering recessive mutations (e.g. i n tumour suppressor genes) or by leading to higher rates o f dosage imbalances o f genes (Knudson, 2001). Approximately 13% o f solid tumours are not aneuploid, but contain mutations that cause a mutator phenotype (Nowak et al., 2002). In colon cancer, the mutator phenotype is caused b y mutations in the mismatch repair genes (e.g. hMLHl  and hMSH2) and is characterized b y  microsatellite instability ( M I N ) . The M I N phenotype and hereditary pre-dispositions to colon cancer arising from mutations i n the mismatch repair genes were discovered based on analysis o f the M I N phenotype i n yeast (Strand et al., 1993; Fishel et al., 1993; Leach et al., 1993; Papadopoulos et at., 1993) and subsequent screening o f human candidate genes based on that work. M I N and C I N phenotypes are thought to increase the chance that multiple genetic changes necessary for tumourigenesis w i l l occur; M I N by causing mutations i n key genes and C I N by L O H and/or dosage imbalance o f genes. A common model from mathematical extrapolations indicates that six to ten genetic alterations are required for cancer (Nowak et al., 2002; Knudson, 2001; Rajagopalan and Lengauer, 2004). These genetic alterations typically confer a growth advantage and become fixed in the population through selection and clonal expansion. After the genetic alteration is fixed in the cell population, the next genetic event would occur i n one o f the cells i n the population, and a similar process o f selection and clonal expansion would occur. Accumulating six to ten o f these genetic events would lead to cancer and M I N and C I N phenotypes could accelerate the rate at which independent "hits" accumulate. There is also evidence linking the amount o f aneuploidy o f a tumour with the severity o f the disease, although using aneuploidy to adjust prognosis and treatment has not been attempted (Rajagopalan and Lengauer, 2004).  3  What can cause this increased rate o f aneuploidy due to increased rates o f chromosome missegregation in tumour cells? Clearly, a very large number o f genes are known that could be responsible and many more are yet to be discovered. Researchers i n recent years have screened for somatic mutations i n candidate C I N genes implicated i n genome stability based on sequence similarity to genes identified i n model organisms such as the budding yeast Saccharomyces cerevisiae.  Mutations in mitotic checkpoint  genes such as hBUBl  and hBUBRl  have been found in colorectal tumour cell lines and  expression o f hBUBl  mutant genes i n n o n - C I N cell lines causes a dominant negative  effect leading to chromosome instability (Cahill et al., 1998). A recent report showed that biallelic mutations i n hBUBIB  were associated with an inherited predisposition to  cancer (Hanks et al., 2004), providing strong support for the model that C I N is a predisposing factor i n cancer initiation/progression. Another spindle assembly checkpoint protein first characterized i n budding yeast, MAD2, has also been found to be transcriptionally silenced i n breast and other cancers ( L i and Benezra, 1996).  MAD2+/-  mice were found to develop lung tumours at a high rate after long latencies, further implicating the mitotic checkpoint in tumourigenesis. In a recent survey screening for mutations i n 100 candidate genes in a panel o f colorectal tumours, the Vogelstein group identified five new C I N genes (MRE11, hZWIO, hZwilch, hRod, Ding (PDS1 /securin homolog) (Wang et al., 2004) mutated i n cancer. These mutations account for <20% o f the colon tumours (Wang et al., 2004) and, therefore, the genetic basis for chromosome instability remains unknown i n the majority o f colon tumours. Finding additional C I N genes i n model organisms i n order to sequence their human homologues as candidate genes i n tumour cell lines w i l l help to identify the complete spectrum o f C I N genes  4  responsible for aneuploidy i n cancer. The success i n identifying the genes responsible for the M I N phenotype and finding mutations in candidate C I N genes provides examples that research with budding yeast can lead to insights i n cancer. Characterizing the genes that are involved i n C I N tumours can provide practical applications i n cancer treatment. Recent experiments have shown that in C I N cell lines that do not have mutations in the spindle assembly checkpoint, introducing a mutation or lowering the expression o f one o f the spindle assembly checkpoint proteins (hMADI)  can  cause massive chromosome loss and non-disjunction, leading to apoptotic cell death i n these aneuploid cancer cell lines (Kops et al., 2004). K n o w i n g the underlying mutations involved i n aneuploidy i n a particular tumour can help us to develop therapies that selectively target the cancer cells for death. For example, finding gene mutations that are synthetic lethal i n budding yeast with the cancer gene mutation that is causing the aneuploidy can help identify candidate proteins i n tumour cell lines that when targeted b y a drug can cause death to the cancer cell without killing adjacent normal cells. In addition, understanding the C I N mutational spectrum can lead to sub-classification o f tumours based on the gene mutated and this could lead to improved diagnostics, prognosis and predictions o f response to different treatments (Rajagopalan and Lengauer, 2004). Finding genes that are involved i n the C I N phenotype in tumour cell lines w i l l provide a rich resource for both clinical and basic research and may also provide targets for gene therapy that w i l l prevent the C I N phenotype i n people pre-disposed to inherited forms o f cancer. M a n y o f the factors that contribute to genomic stability i n mitosis are also important during meiosis and understanding their function could lead to insights into the  5  generation o f trisomies i n humans such as trisomy 21 in D o w n Syndrome. N o n disjunction events leading to aneuploidy i n the first meiotic division i n female oocytes account for the majority o f trisomies (Hassold and Sherman, 2000). Trisomies occur i n approximately 0.3% to 0.5% o f live births (Antonarakis et al., 2004) and a maternal age effect is associated with trisomies (Reeves et al., 2001). A two hit hypothesis has emerged to explain the dynamics o f trisomy generation. Defects i n the spindle assembly checkpoint or sister chromatid cohesion have been postulated to be one o f the hits required for generating trisomies. The generation o f bivalents that are achiasmatic (homologous chromosomes that do not undergo a recombination event and do not have chiasmata) or have a chiasma located too far or too near to centromeres can lead to an increased chance o f non-disjunction and is postulated to be the other hit required for generating trisomies. The combination o f these two hits contributes to non-disjunction when the first meiotic division is completed shortly before ovulation (Hassold and Sherman, 2000). Investigating factors involved i n chromosome segregation i n mitotic cells and investigating their roles i n meiosis could lead to understanding the mechanisms o f trisomy generation.  Budding yeast as a model organism Saccharomyces  cerevisiae, a budding yeast, has served as a model organism for  understanding essential and important processes i n the mitotic cell cycle. Components and pathways involved i n basic intracellular processes such as D N A replication, cell cycle checkpoints, D N A repair, sister chromatid cohesion, kinetochore function and cell cycle machinery are conserved from yeast to man (Kitagawa and Hieter, 2001).  6  Saccharomyces  cerevisiae is a unicellular eukaryote that has both a haploid and a diploid  life cycle. 2n diploids can be induced to sporulate by nitrogen starvation on a poor carbon source and can form four I n haploid spores that are encased i n a durable spore wall. The spores can be germinated and each o f the haploid offspring can be recovered, making budding yeast especially useful for genetic manipulation. A s well, haploid yeast cells o f opposite mating types can mate and form diploids, and following meiosis, hybrid spore products can be detected and analyzed with novel combinations o f mutations (reviewed i n Rose et al., 1990). One o f the greatest assets in using yeast as a model organism is the ease in which the genome can be manipulated, i.e. recombinant genetics through homologous recombination. Deletions, point mutations, and epitope tagged versions o f genes can be introduced at their endogenous loci facilitating genetic, phenotypic, cell biological, and biochemical characterization i n cells (Longtine et al., 1998). Budding yeast was used to identify many o f the key components o f the cell cycle machinery which are highly conserved i n eukaryotic cells. Morphology o f individual yeast cells can be correlated with a particular stage o f the cell cycle as shown i n Figure 1. Unbudded cells are i n G l and have recently exited mitosis and undergone cytokinesis. Small budded cells are entering S phase and undergoing spindle pole body duplication (site o f microtubule nucleation, similar to the centrosome in human cells). A s the bud grows larger, the yeast cell passes through S phase into G 2 . Large budded cells are generally classified as G 2 / M cells and are approaching anaphase. This correlation between morphology and the cell cycle was used to isolate mutants that arrested budding yeast cells at particular stages i n the cell cycle. They helped to isolate proteins that were  7  Interphase chromatin  I  Kinetochore  0  Nucleus  •  Cohesin  -•  Mitotic Spindle  0  Spindle Pole Body  Chromatid  Metaphase to anaphase transition cohesin dissociated and cohesion destroyed  DNA replication Cohesion establishment Mono-polar attachment i-X)  Bi-polar attachment  Figure 1-1 - The budding yeast cell and chromosome cycle. Cellular morphology can be correlated to cell cycle progression. The chromosome cycle (condensation, replication, sister chromatid cohesion and separation) is shown with cell cycle progression.  8  important for cell cycle progression in budding yeast, and b y homology, other eukaryotes (Hartwell, 1980; W o o d and Hartwell, 1982). D r . Leland Hartwell w o n the N o b e l Prize i n Physiology and Medicine in 2001 for his pioneering work on "key regulators o f the cell cycle". Budding yeast was the first eukaryote to have its genome sequenced (Goffeau et al., 1996), and many technologies, such as micro-array analysis o f gene expression, S A G E (Serial Analysis o f Gene Expression) (Velculescu et al., 1997), and S G A (Synthetic Genetic Array) (Tong et al., 2001) screens were first developed i n budding yeast. The experimental tractability o f budding yeast facilitates the testing o f the validity o f novel genomic and proteomic data sets. There are - 6 0 0 0 genes i n budding yeast, - 1 1 0 0 o f which are essential for viability; this has led to the generation o f gene deletion sets that contain deletion mutants o f the non-essential genes (the haploid sets and the homozygous diploid set) as well as all the genes in yeast (the heterozygous diploid deletion set) (Giaever et al., 2002). In studying factors that are important for chromosome segregation fidelity, there are several differences that should be pointed out between budding yeast and mammalian cells. Budding yeast chromosomes and centromere D N A are much smaller than their mammalian counterparts. The average yeast chromosome is - 1 M e g a base pairs (Mbp), and the centromere is approximately 125 base pairs (bp), whereas human chromosomes are approximately 150 M b p and centromeres i n human cells can be as large as 5 M b p . (Loidl, 2003). One microtubule is bound to each yeast kinetochore whereas dozens o f microtubules are attached to human kinetochores. Budding yeast kinetochores remain bound to the spindle pole body through interphase and tend to cluster together (Jin et al.,  9  2000) and microtubules bind kinetochores soon after replication. In human cells microtubules are not connected to kinetochores immediately after replication (Gadde and Heald, 2004). Budding yeast undergoes a "closed mitosis" i n which the nuclear envelope does not breakdown during the course o f mitosis (Loidl, 2003), and the spindle pole bodies remain anchored i n the nuclear membrane during mitosis. In contrast, mammalian cells undergo an "open mitosis" i n which the nuclear envelope breaks down and chromosomes and centrosomes become part o f the cytoplasm.  The Cell Cycle and Genome Stability Genome stability in dividing cells is intimately tied to cell cycle control. C e l l cycle control is crucial to ensure that events during the cell cycle take place with high fidelity (for a review see Murray and Hunt (1993)). This involves ensuring that certain events happen only after other events have occurred (e.g. sister chromatids cannot be segregated until they have been duplicated and until they are attached to opposite spindle pole bodies) and that certain events can only happen once during the cell cycle (e.g. the firing o f origins o f replication, the duplication o f the spindle pole body). There are points during the cell cycle at which cells w i l l commit to completing events. For example, the Start point i n the budding yeast cell cycle is the point i n G l when the cell begins the vegetative cell cycle and cannot thereafter be affected by mating pheromone. Another example w o u l d be the metaphase to anaphase transition when sister chromatids are pulled apart. A breakdown i n cell cycle control could lead to errors that contribute to chromosome missegregation and aneuploidy. T w o C I N genes mutated i n colorectal  10  tumours are hCDC4 (Rajagopalan et al., 2004) and DING/SECURIN  (Wang et al., 2004);  two genes important for cell cycle control. The cell cycle control machinery is conserved among all eukaryotes. C y c l i n Dependent Kinases ( C D K s ) serve as the regulators o f the cell cycle and, i n concert with a bound cyclin subunit (which serves to activate its kinase activity), can drive the cell cycle through phosphorylating substrates. Cyclins were first discovered to be proteins that accumulated and then disappeared i n a cell cycle dependent manner (Evans et al., 1983; Cross, 1988; Hadwiger et al., 1989; Nash et al., 1988; Murray, 1989). This periodic accumulation and degradation allows C D K activity to be regulated i n a periodic manner and drives the cell cycle through phosphorylating substrates at the right time during the cell cycle. C D K s can also be regulated b y phosphorylation and the binding o f inhibitors ( C D K Inhibitors, C K I s ) such as S i c l p i n budding yeast. Cdc28p and Pho85p are the C D K s involved i n cell cycle progression i n budding yeast w i t h Cdc28p being the main regulator (Mendenhall and Hodge, 1998). Cdc28p is expressed and maintained at a high level throughout the cell cycle and cyclin binding  t  controls its activity. In budding yeast, G l cyclins ( C l n l p , Cln2p and Cln3p) promote events after mitotic exit and cytokinesis and are important for passage through Start during G l (Cross, 1990; Hadwiger et al., 1989; Richardson et al., 1989). S phase cyclins (Clb5p, Clb6p, Clb3p, and Clb4p) promote entry into S phase (Dahmann et al., 1995; Richardson et al., 1990). Mitotic cyclins ( C l b l p , Clb2p) drive the cell cycle through mitosis and past the metaphase to anaphase transition (Fitch et al., 1992; Ghiara et al, 1991).  11  Coordinated expression and translation o f m R N A s and proteins during the cell cycle is controlled b y phosphorylation o f transcription factors b y C D K s (Wittenberg and Reed, 2005). These proteins are important for events that are coordinated i n a cell cycle dependent manner such as bud morphogenesis, spore wall deposition, and D N A duplication. Proper cell cycle control is also dependent on protein degradation by regulated proteolysis (Vodermaier, 2004). For example, S i c l p is a C K I that must be degraded during G l in order for the G l / S phase transition to occur (Schwob et al., 1994; Donovan et al., 1994; B a i et al., 1996). Proteolysis o f target proteins is a mechanism for cells to proceed through a "gate" i n the cell cycle whereby the cell cannot return to an earlier stage o f the cell cycle, thus preventing events from re-occurring that must occur only once i n the cell cycle such as D N A replication or spindle pole body duplication. M u c h o f the cell cycle regulated proteolysis is accomplished by targeting proteins for degradation b y the 26S proteosome (Hershko, 1997; Chun et a l , 1996). This targeting is accomplished by attaching ubiquitin, a highly conserved polypeptide, onto the lysine residues o f target proteins. Ubiquitin on proteins is then extended and this polyubiquitin chain serves as a tag for degradation by the 26S proteosome. Ubiquitin mediated proteolysis is well conserved i n eukaryotes and the process o f attaching ubiquitin to target proteins is accomplished by a well conserved cascade o f enzymes. E l ubiquitin activating enzymes bind to ubiquitin and prepare the moiety for subsequent binding to an E 2 , a ubiquitin conjugating enzyme (or Ubiquitin Carrying Enzyme, U B C ) . A n E 3 complex, ubiquitin ligase, w i l l bring substrate proteins and the U B C - u b i q u i t i n together and E 3 complexes provide the specificity i n the targeting o f proteins.  12  The Anaphase Promoting Complex / Cyclosome ( A P C / C ) and the S C F complex ( S k p l p / C u l l i n / F-box protein) are E3 complexes that are important for cell cycle proteolysis (for reviews see Peters, 1998; Vodermaier, 2004; Page and Hieter, 1999; Willems et al., 1999; Zachariae and Nasmyth, 1999). The two complexes each have a cullin family protein (Apc2p i n the A P C / C and Cdc53p i n the S C F ) that acts as a scaffold and structural protein aiding i n E 2 recruitment (Ohta et al., 1999; Tang et al., 2001; Zheng et al., 2002). Both complexes depend on a R I N G finger protein ( A p c l l p i n the A P C / C and R b x l p in the S C F complex) for U B C - u b i q u i t i n recruitment (Ohta et al., 1999; Leverson et al., 2000; Gmachl et al., 2000). The complexes contain multiple subunits and they depend on substrate specificity factors that can bind onto the core A P C / C or S C F complex and target specific proteins for degradation. In the case o f the A P C / C the specificity factors are Cdc20p and C d h l p (Visintin et al., 1997) and a meiosis specific factor, A m a l p (Cooper et al., 2000), and for the S C F complex the specificity factors are the different F-box proteins that bind the S C F (e.g. Cdc4p, G r r l p , Met30p) (Skowyra et al., 1997). The specificity factors contain protein-protein interaction domains such as W D - 4 0 repeats, which w i l l contribute to protein-protein binding and are responsible for bringing substrates together with the UBC-ubiquitin. The A P C / C (Cdc20p) is responsible for targeting proteins that are important for the metaphase to anaphase transition (such as P d s l p , discussed below) and also passage through mitosis. A P C / C ( C d h l p ) is responsible for proteolysis later i n mitosis and during early G l and targets substrates such as the mitotic cyclin, Clb5p, as well as Cdc20p (Visintin et al., 1997; Pfleger et al., 2001). The S C F is active at many stages o f the cell cycle and has proteolytic functions unrelated to cell cycle progression but it is especially important for  13  the G l / S phase transition, with one o f its main substrates being the C K I S i c l p (Bai et al., 1996). The S C F complex also targets the G l cyclins ( C l n l p and Cln2p) and Cdc6p, a protein involved i n pre-replication complex formation (discussed below) for degradation (Willems et al., 1999).  DNA Replication and Genome Stability Eukaryotic genomes are much larger than prokaryotic genomes and i n order to replicate the entire genome i n the time frame o f the typical cell cycle, multiple origins o f replication are needed (for reviews see B e l l and Dutta, 2002; Diffley, 2004). Origins o f replication are chromosomal elements that are necessary to initiate D N A replication and establish bi-directional replication forks (Bell, 1995). Origins o f replication direct the binding o f the pre-Replicative Complex (pre-RC) onto D N A and subsequent loading o f the other proteins involved at replication forks (e.g. D N A polymerases, processivity factors such as Proliferating C e l l Nuclear Antigen, P C N A ) (Bell and Dutta, 2002). In budding yeast, these origins o f replication are approximately 100 to 150 base pairs and include highly conserved A elements and less w e l l conserved B elements that may aid i n unwinding o f the D N A double helix (Bell, 1995). The sequence o f origins o f replication in other eukaryotes is less well established (Bielinksky and Gerbi, 2001; B l o w et al. 2001) and the surrounding chromatin domain appears to be important. The pre-RC is assembled onto chromosomes and directs the placement o f factors that w i l l be used to replicate D N A . The pre-RC consists o f the Origin Recognition Complex ( O R C , made up o f O r e l - 6), Cdc6p, C d t l p and M c m 2 - 7 complex. O R C contains D N A binding motifs and interacts directly with origins o f replication. In  14  budding yeast the binding o f O R C to D N A is constitutive and does not appear to be cell cycle regulated (Ogawa et al., 1999; Aparicio et al., 1997; Santocanale and Diffley, 1996; Tanaka et al., 1997). Cdc6p requires O R C to bind D N A (Neuwald et al., 1999; Coleman et al., 1996) and is a substrate o f the S C F (Cdc4p) and is targeted for degradation i n late G l and early S phase (Drury et al., 1997; Elsasser et al., 1999; Perkins et al., 2001; Piatti et al., 1996). Cdc6p is required for the loading o f the M c m 2 - 7 complex and Cdc6p degradation (which is dependent on its phosphorylation by C D K ) is one mechanism to ensure that D N A is not re-replicated (Coleman et al., 1996). The M c m 2 - 7 complex binds chromatin i n a Cdc6p and O R C dependent manner and this binding o f M c m 2 - 7 may be one o f the key functions o f Cdc6p and O R C (Hua and Newport., 1998). M c m 2 7 is regulated also i n a cell cycle manner but not by proteolysis. Phosphorylation o f subunits i n the M c m 2 - 7 complex that are not chromatin bound targets the complex for export from the nucleus to the cytoplasm (Labib et al., 1999; Nguyen et al., 2000; Pasion and Forsburg, 1999). The pre-RC may exist to provide a chromatin framework for binding o f the subsequent proteins that are needed for D N A replication and to provide a mechanism for establishing origins o f replication that w i l l replicate only once i n the cell cycle (Bell and Dutta, 2002). There is some evidence that M c m 2 - 7 functions as a D N A helicase but this has not been completely proven ( Y o u et al., 2002; Lee and Hurwitz, 2002). Once the pre-RC is formed at origins o f replication i n late G l , high levels o f C D K activity w i l l also induce the subsequent binding o f other proteins that w i l l begin D N A replication. Another kinase, Cdc7p / Dbf4p (Dbf4p Dependent Kinase, D D K ) , is also needed along with C D K to initiate S phase (Pasero et al., 1999). M c m l O p and Cdc45p  15  are loaded onto the origins o f replication b y the pre-RC and direct the initiation o f replication b y recruiting other proteins such as replication protein A ( R P A ) , a single stranded D N A ( s s D N A ) binding protein that protects and stabilizes s s D N A generated b y origin firing, and also the D N A polymerase a that is needed to generate the R N A primers required to initiate D N A replication (Bell and Dutta, 2002). Once the R N A primers are generated and D N A polymerase a extends the fragment by 20 bps, the Replication Factor C complex ( R F C , consisting o f R f c l - 5) displaces the polymerase, and acts to load Proliferating C e l l Nuclear Antigen ( P C N A ) , a ring like "clamp" that encircles D N A and provides processivity to D N A polymerase 5, which replaces D N A polymerase a , and acts as the main D N A polymerase that replicates D N A (Majka and Burgers, 2004; Hubscher et al., 2002). This polymerase switch between the primase and the main processing polymerase is not w e l l understood. Proper timing and control o f D N A replication plays an important role i n genome stability. Mis-regulation o f origins o f replication can lead to increased genomic instability (Lengronne and Schwob, 2002; Huang and Koshland, 2003; Kolodner et al., 2002). During D N A replication, lesions that are either spontaneously generated or caused b y D N A damaging agents can lead to the replication fork being stalled, broken, or displaced from D N A (Longhese et al., 2003). The stabilization o f replication forks and processing and repair o f D N A lesions are important in maintaining genome stability. The checkpoint mechanisms and repair processes w i l l be discussed i n sections below.  16  The Mitotic Spindle and Chromosome Segregation The mitotic spindle is the specialized structure assembled during mitosis i n order to physically pull apart sister chromatids during anaphase (Gadde and Heald, 2004). It is composed o f microtubules, one o f the key components o f the cytoskeleton providing structure to cells. Microtubules are non-covalent polymers o f tubulin. The tubulin subunits o f microtubules are made up o f a hetero-dimer o f a-tubulin ( T u b l p and Tub3p in budding yeast) and P-tubulin (Tub2p i n budding yeast), two proteins that share approximately 50% identity (Desai and Mitchison, 1997). The hetero-dimers o f a tubulin and P-tubulin are arranged i n a head to tail conformation giving the microtubule structural polarity. Microtubule plus ends are more dynamic and form the growing head o f the structure, while their minus ends remain anchored and nucleated at microtubule organizing centres (centrosomes i n human cells and spindle pole bodies i n budding yeast). The head to tail interactions o f the hetero-dimers are arranged into linear protofilaments that w i l l associate to form a hollow cylindrical tube o f a microtubule (Kline-Smith and Walczak, 2004). Microtubules are dynamic structures and the prevailing model for their action is that o f dynamic instability driven by four factors: 1) the rate o f polymerization (growth) 2) the rate o f de-polymerization (shrinkage) 3) the frequency and occurrence o f catastrophes (the change from growth to shrinkage) 4) the frequency and occurrence o f rescues (the change from shrinkage to growth) (Gadde and Heald, 2004; Desai and Mitchison, 1997; Kline-Smith and Walczak, 2004). While both a and P-tubulin can bind G T P , G T P bound P-tubulin is more important in terms o f regulation and dynamic instability. G T P bound P-tubulin is incorporated at the growing ends o f microtubules  17  during growth. During or shortly after polymerization, the G T P bound form o f p-tubulin is hydrolyzed and this G D P bound form o f P-tubulin does not exchange and forms the majority o f the P-tubulin in the microtubule complex. There is thought to be a lag between incorporating the G T P bound tubulin and G T P hydrolysis, and this lag provides a G T P cap on growing ends o f microtubules that is thought to provide some stability to the growing plus end. When a catastrophe event occurs, de-polymerizing G D P bound Ptubulin is released rapidly and the plus end undergoes rapid shrinkage. G T P is exchanged for G D P on the free P-tubulin, allowing this subunit to undergo polymerization when a rescue event occurs. This dynamic instability is thought to allow rapid re-organization o f microtubules and allow a process such as "search and capture" (Kline-Smith and Walczak, 2004). The mitotic spindle i n budding yeast consists o f microtubules that emanate from spindle pole bodies that either bind to kinetochores (kinetochore microtubules), or that form the central spindle where microtubules from the opposite spindle pole bodies interact i n an anti-parallel array (interpolar microtubules). The spindle pole body is also the site o f nucleation o f the astral microtubules that w i l l project out into the cytoplasm. Microtubules are dynamic and unstable but microtubule associated proteins as well as attachment to kinetochores can stabilize microtubules (Saxton et al., 1984; Zhai et al., 1996). Kinetochore-microtubule attachments, once attached to opposite spindle pole bodies and stabilized, provide the signal for the metaphase to anaphase transition to occur. The mechanism by which microtubules w i l l bind to kinetochores is termed "search and capture" and consists o f microtubules at the plus end alternately growing and  18  shrinking, probing for kinetochores. Once bound, the kinetochore microtubule attachments are stable. Because o f sister chromatid cohesion the sister kinetochores are oriented away from each other allowing attachments o f the sister kinetochores to occur with microtubules from opposite spindle poles (Tanaka et a l , 2000). Kinetochore regulating factors such as Ipllp/Aurora Kinase (discussed below) w i l l destabilize kinetochore microtubule attachments that are mono-polar (Biggins and Murray, 2001; Dewar et al., 2004; Pinsky et al., 2003; Tanaka et al., 2002). Tension that is produced b y bi-oriented sister kinetochore microtubule attachments are thought to further stabilize •these attachments (Kline-Smith et al., 2005). Microtubules in the absence o f spindle poles and kinetochores have also been shown to self assemble into spindles (Heald et al., 1996) and mechanisms in addition to "search and capture" may be at work i n mitotic spindle formation. Once sister chromatids have formed stable kinetochore microtubule attachments (bi-oriented to opposite spindle poles), a process that is monitored b y the spindle assembly checkpoint (discussed below), sister chromatid cohesion is dissolved and the mitotic spindle pulls sister chromatids towards the poles. This segregation o f sister chromatids is dependent on microtubule dynamics and also microtubule motor proteins. Microtubule de-polymerization can serve to drag sister chromatids towards the poles b y either o f two mechanisms. One mechanism is that kinetochores induce microtubule disassembly at plus ends while maintaining their attachments, "chewing" their way towards the spindle pole. The second mechanism is kinetochore microtubule attachment and microtubule stability at the plus end remaining unaffected, and an increased rate o f de-polymerization occurs at the minus end, pulling the chromosomes to the poles (Gadde  19  and Heald, 2004). Microtubule motor proteins are also involved i n generating force that can pull chromosomes apart. Microtubule motor proteins exist either as microtubule minus end or microtubule plus end directed motors. One example is the kinesin Kar3p, which is a minus end directed motor that can increase de-polymerization o f microtubules at the minus end (Desai and Mitchison, 1997; Biggins and Walczak, 2003). Movement o f chromosomes towards poles represents anaphase A , and after this movement, poles w i l l separate by elongation o f the mitotic spindle (anaphase B ) , a process that is also driven by stabilization o f microtubule dynamics. Finally, before cytokinesis, the mitotic spindle must be disassembled and this disassembly is driven b y de-polymerization (Seshan and A n i o n , 2004; Cheeseman and Desai, 2004).  The Centromere and Kinetochore in Budding Yeast The kinetochore, which consists o f the centromere D N A and associated proteins, is essential as an attachment point for microtubules emanating from opposite spindle pole bodies and also as a platform for sensing proper bi-polar attachment. The budding yeast centromere is a "point" centromere and consists o f three conserved elements ( C D E I, C D E II, and C D E III) that span 125 base pairs (Loidl, 2003). The associated kinetochore proteins have been categorized into inner kinetochore proteins that have direct connections with the centromere D N A (or are i n a complex with proteins that directly interact with C E N D N A ) , central kinetochore proteins that serve as scaffolding and link the inner kinetochore to the outer kinetochore proteins, and outer kinetochore proteins that link the kinetochore to microtubules (for reviews see Cheeseman et al., 2002; Measday and Hieter, 2004; M c A i n s h et al., 2003; Skibbens and Hieter, 1998; Kitagawa  20  and Hieter, 2001). The budding yeast kinetochore is assembled i n a hierarchical manner with distinct sub-complexes (De W u l f et al., 2003). The budding yeast inner kinetochore consists o f a complex called C B F 3 (which is made up o f N d c l O p , C t f l 3 p , Cep3p and S k p l p ) that binds to the C D E I I I region o f the centromere. A l l o f the components o f the C B F 3 complex are essential for viability. C b f l p protein is also at the yeast inner kinetochore and it binds at the C D E I region o f the centromere. C b f l p is not essential for viability. Cse4p is a well conserved histone H 3 variant that replaces H 3 i n the histone octamer in and around the centromere region and is essential for viability (Cheeseman et al., 2002). Within the C B F 3 complex, only S k p l p is w e l l conserved from yeast to man (Kitagawa and Hieter, 2001) and the role o f human S k p l p at the kinetochore is unclear (Gstaiger et al., 1999). The lack o f conservation o f the other members o f the C B F 3 complex may reflect the difference i n centromere sequence between yeast and humans. S k p l p is an especially interesting protein as it plays roles i n two distinct processes. S k p l p was originally identified i n our laboratory as a high copy suppressor o f a temperature sensitive mutant, ctf 13-30 (Connelly and Hieter, 1996), and characterized as a component o f the kinetochore. S k p l p was also found as a suppressor o f cdc4 mutants (Bai et al., 1996) and characterized as a subunit o f the S C F . F-box proteins bind to the S C F through S k p l p and their F-box domains; C t f i 3 p is also an F-box protein and binding o f S k p l p to C t f l 3 p requires C t f l 3 p ' s F-box domain (Russell et al., 1999). Temperature sensitive alleles o f SKP1 fall into two different classes; mutants that arrest at the restrictive temperature with a G l / S profile with a defect i n S C F function, e.g. skpl3, and mutants that arrest at the restrictive temperature with a G 2 / M profile and a defect  21  i n kinetochore function, e.g. skpl-4 (Connelly and Hieter, 1996). SGT1 was identified as a high copy suppressor o f skpl-4 (Kitagawa et al., 1999) and S g t l p was found to be a protein that directly interacted with S k p l p and functioned with S k p l p at the kinetochore and the S C F . SGT1 is an essential gene and temperature sensitive alleles o f SGT1 can be distinguished i n the same way as SKP1 temperature sensitive alleles can, into mutants with an S C F defect (sgtl-5) or mutants with a kinetochore defect (sgtl-3). A t the kinetochore, S g t l p interaction with S k p l p is mediated by the H S P 9 0 chaperones and their activity is required to activate C t f l 3 p and promote C B F 3 assembly (Bansal et al., 2004; Lingelbach and Kaplan, 2004). The central kinetochore consists o f at least 3 different sub-complexes, the N D C 8 0 complex, the M E N D complex and the C O M A complex. There are also other proteins o f the central kinetochore that have been isolated that have not been placed into these subcomplexes but together they form a large macro-molecular complex at the central kinetochore that can be co-purified by biochemical assays such as coimmunoprecipitation (Pot et al., 2003; Measday et al., 2002; D e W u l f et al., 2003; Measday and Hieter, 2004). A l l o f the central kinetochore proteins depend on the C B F 3 inner kinetochore complex for localization to the kinetochore but the question o f whether or not these sub-complexes interact with the inner kinetochore independently o f each other or whether there is a higher level o f regulation and interdependency is still unknown (Measday and Hieter, 2004). M a n y o f the proteins o f the central kinetochore are not essential for viability but the corresponding deletion mutants lose chromosomes at much higher rates than w i l d type strains.  22  The outer kinetochore consists o f proteins that bind to microtubules. Members o f the D A M 1 complex have been shown to bind the mitotic spindle and D a m l p has been shown to bind microtubules in vitro (Kang et al., 2001; L i et al., 2002; Cheeseman et al., 2002). The localization o f the D A M 1 complex to the kinetochore is dependent on an intact mitotic spindle. Recent work has shown that the D A M 1 complex forms a ring around microtubules (Miranda et al., 2005; Westermann et al., 2005). There are also other proteins that have been localized to the kinetochore with microtubule binding capabilities, such as Stu2p, B i k l p , B i m l p and kinesin related proteins (microtubule motors) Cin8p, K i p l p and K i p 3 p (He et al., 2001; Cheeseman et al., 2002). Researchers have also begun to investigate proteins that regulate the budding yeast kinetochore. Sister kinetochores initially are attached i n a mono-polar manner to the old S P B . Kinetochore microtubule capture and release is not w e l l understood but the mechanisms for regulating proper bi-polar attachment have begun to be elucidated. The current model involves a kinase complex consisting o f S l i l 5 p / I p l l p that senses the lack o f tension generated b y mono-polar attachment and phosphorylates D a m l p , Ndc80p and N d c l O p (Biggins and Murray, 2001; Dewar et al., 2004; Pinsky et al., 2003; Tanaka et al., 2002). This phosphorylation is hypothesized to remove the kinetochore-microtubule attachment, and new attachments would have to be established. A s w e l l as this mechanism to ensure bi-polar attachment, the spindle assembly checkpoint, w h i c h i n budding yeast consists o f M p s l p , M a d l p , Mad2p, Mad3p, B u b l p and Bub3p, generates a wait anaphase signal that inhibits the metaphase to anaphase transition until all sister chromatids have achieved bi-polar attachment (discussed below).  23  Sister Chromatid Cohesion Eukaryotic chromosome replication is separated i n time from chromosome segregation. After S phase each chromosome w i l l consist o f two identical sister chromatids and i n order to ensure that sister chromatids segregate equally into separate cells during mitosis, they must be kept attached until mitosis. This attachment, or sister chromatid cohesion, ensures that sister chromatids stay together and promotes b i orientation o f the sister chromatids such that amphitelic attachments (sister kinetochores binding to microtubules from opposite spindle pole bodies) can occur (Tanaka et al., 2000). Defective sister chromatid cohesion or defective regulation o f cohesion can lead to precocious sister chromatid separation leading to chromosome loss or non-disjunction. The mechanism o f sister chromatid cohesion is w e l l conserved i n eukaryotes and depends on a chromosomal protein complex called cohesin (for reviews see Nasymth, 2001; Uhlmann, 2003; Uhlmann, 2004; Cohen-Fix, 2001). Cohesin is a complex made up o f at least four subunits, a heterodimer o f two S M C (Structural Maintenance o f Chromosomes) proteins, S m c l p and Smc3p, bound to two other proteins, S c c l p and Scc3p (Michaelis et al., 1997; Guacci et al., 1997; Losada et al., 1998). Structural studies have shown that cohesin is a ring link structure (Anderson et al., 2002; Haering et al., 2002; Gruber et al., 2003). S m c l p and Smc3p are dimerized i n a head to head and a tail to tail conformation with the circumference o f the cohesin ring consisting o f the flexible coiled coil regions o f S m c l p and Smc3p. The tails o f the S m c l p and Smc3p interact to form a hinge region (see Figure 2 for a model). The head regions o f S m c l p and Smc3p have similarity to the A T P a s e domains o f A B C transporters and it is thought that their binding is dependent on A T P binding. S c c l p binds the head region o f S m c l p and  24  cohesin  heads coiled coil Smc3  Smc1  From Weitzer et al., 2003  hinge  Figure 1-2 - The cohesin complex forms a ring like structure. The coiled coil regions o f S m c l p and Smc3p form the circumference o f the ring, with their tails forming a hinge region and their heads associating to close the ring. S c c l p binds to the head region with Scc3p associating with S m c l p and Smc3p through S c c l p .  Smc3p, w i t h Scc3p binding to S c c l p (Haering et al., 2002). S c c l p can bind both A T P and A D P bound forms o f S m c l p and Smc3p, while A D P bound S m c l p and Smc3p heads bind each other with less affinity than A T P bound S m c l p and Smc3p. A T P hydrolysis by S m c l p and Smc3p is needed for cohesin binding to D N A which supports the idea that A T P bound S m c l p and Smc3p w i l l hydrolyze A T P and bind D N A and then have the A D P bound S m c l p and Smc3p heads stabilized by S c c l p and Scc3p (Arumugam et al., 2003; Weitzer et al., 2003). A l l four subunits o f the cohesin complex are essential for viability in budding yeast (Saccharomyces Genome Database, S G D ) . The cohesin complex associates with discrete chromosomal regions prior to S phase and w i l l stay bound to chromatin until its dissolution at the metaphase to anaphase transition (Michaelis et al., 1997). There is no consensus sequence o f D N A that cohesin w i l l bind to, although preferred cohesin binding sites exist. The centromere is the site o f the most deposition o f cohesin (Blat and Kleckner, 1999; Tanaka et al., 1999; Megee et al., 1999) and along the chromosome arms cohesin deposition generally occurs in intergenic regions with high A T content. Cohesion attachment sites are spaced roughly  25  10 to 15 kbases apart (Blat and Kleckner, 1999; Tanaka et al., 1999). The cohesin complex can be moved along chromatin and appears to localize to sites o f convergent transcription (Glynn et al., 2004; Lengronne et al., 2004). Cohesin is loaded onto unreplicated chromatin by a protein complex consisting o f Scc2p and Scc4p (both o f which are essential for viability i n budding yeast) (Ciosk et al., 2000) and this loading occurs i n late G l . During D N A replication, sister chromatid cohesion w i l l be established using the cohesin that has been loaded. The establishment o f sister chromatid cohesion is not w e l l understood but is known to require replication and the acetyltransferase activity o f E c o l p / Ctf7p (Skibbens et al., 1999; Toth et al., 1999). The proteinaceous ring structure o f cohesin has given rise to the idea that cohesin encircles the two sister chromatids and that the connection between the sister chromatids is a topological one (Campbell and Cohen-Fix, 2002). The replication forks may slide through the cohesin complex and leave both sister chromatids circled b y the cohesin complex. There is no clear structure that has been isolated showing a cohesin ring encircling sister chromatids and the idea that cohesin forms a ring around D N A remains unproven. Cohesin could form the "glue" that sisters bind to i n order to remain associated with one another (Huang et al., 2005; Campbell and CohenF i x , 2002). Sister chromatid cohesion must be maintained through G 2 and into mitosis when all sister kinetochores have achieved bipolar attachment to the mitotic spindle. W h e n bipolar attachment occurs cohesion must be dissolved between the sister chromatids i n order to allow the sister chromatids to be pulled apart. This dissolution o f sister chromatid cohesion is dependent on the cleavage o f the S c c l p subunit o f cohesin by  26  Separase (Uhlmann et al., 2000), and triggers the metaphase to anaphase transition. The Separase, E s p l p i n budding yeast, is tightly regulated by a protein called Securin, P d s l p in budding yeast, i n order to ensure that S c c l p cleavage and the dissolving o f sister chromatid cohesion does not occur too early (reviewed i n Yanagida, 2000). Securin is a protease inhibitor and binds to Separase to inhibit its activity. P d s l p is a target o f the A P C / C and its targeted degradation and subsequent activation o f E s p l p is checkpoint dependent (discussed below). Inhibition o f the A P C / C (Cdc20p) induced b y D N A damage or improper attachment o f mitotic spindles and kinetochores can lead to high P d s l p levels that w i l l generate a wait anaphase signal. In unperturbed cell cycles, once all sister kinetochores are properly attached, P d s l p is ubiquitinated by the A P C and is degraded by the 26S proteosome. Its degradation triggers E s p l p protease activity, which w i l l then cleave S c c l p leading to sister chromatid segregation. P d s l p is also required for the nuclear localization o f E s p l p and in that manner also plays a role i n activating Separase (Jensen et al., 2001). S c c l ' s cleavage by E s p l p releases the cohesin complex from sister chromatids by making an opening in the cohesin ring allowing the cohesin complex to dissociate from chromatin (Uhlmann et al., 2000). A cleavage product o f S c c l p also prevents the association o f S m c l p and Smc3p's head regions, providing another mechanism to ensure that sister chromatid cohesion is eliminated rapidly (Weitzer et al., 2003). This cleavage product is subsequently degraded i n order not to interfere with cohesin i n the next cell cycle (Rao et al., 2001). After mitotic exit and cytokinesis, the new daughter cells i n G l w i l l begin preparing for a new cycle o f sister chromatid cohesion b y forming the S m c l p / Smc3p heterodimer i n anticipation o f S c c l p ' s transcription and translation in late G l (Uhlmann, 2004).  27  One important difference exists between sister chromatid cohesion i n budding yeast and sister chromatid cohesion in human cells. In budding yeast, the cohesin complex remains associated w i t h centromeres and chromosome arms until anaphase whereas i n human cells 95% o f the cohesin (along the chromosome arms) is released during prophase (Morrison et al., 2003). Cohesin released during prophase is not dependent on S c c l p cleavage and the cohesin complex remains largely intact after being released from the chromosome arms. This release is dependent on the mitotic kinase, Polo like kinase (Polo, Cdc5p i n budding yeast) but the mechanism for this release and why centromeric cohesion is unaffected is unclear (Sumara et al., 2002).  Additional proteins required for sister chromatid cohesion There are other proteins that are required for sister chromatid cohesion. Pds5p is an essential protein that binds to the same chromosomal locations as the cohesin complex. PDS5 mutants can establish cohesion but exhibit precocious sister chromatid separation and have a maintenance o f cohesion defect (Hartman et al., 2000; Panizza et al., 2000). Pds5p is sumoylated ( S U M O is a small protein conjugate similar to ubiquitin) and its sumoylation is required for the efficient dissolution o f sister chromatid cohesion (Stead et al., 2003). Characterization and isolation o f the proteins involved i n sister chromatid cohesion has employed a precocious sister chromatid separation assay (Guacci et al., 1994; Straight et al., 1996; Michaelis et al., 1997). This assay measured the percentage o f cells that had precociously separated sister chromatids after D N A replication as measured in cells arrested in G 2 / M by a microtubule depolymerizing agent. W i t h no  28  microtubules to pull sister chromatids apart, w i l d type cells should have sister chromatids in close proximity, whereas cells with defective cohesion show precocious separation o f sister chromatids. In the most commonly used approach, budding yeast strains are constructed with tetracycline (TET) or lactose ( L A C ) operator sequence arrays integrated at different chromosome locations. Green Fluorescent Protein (GFP) tagged T E T or L A C repressor proteins are expressed i n these strains and visualization o f the G F P signal is used to assess the proximity o f sister chromatids containing the operator arrays. Defects i n sister chromatid cohesion increase the percentage o f cells that exhibit two G F P signals (indicating that there was precocious sister chromatid separation) as opposed to one signal (no precocious separation). Mutations i n genes coding for subunits o f the cohesin complex (SCC1, SCC3, SMC1, SMC3), or i n the cohesin loading complex (SCC2, SCC4), caused a high increase i n the percentage o f cells that exhibit two G F P signals (up to 60% o f mutant cells exhibited 2 G F P signals compared to 8% to 10% i n w i l d type cells). A n increasing number o f non-essential gene deletion mutants (strains that contain a deletion o f a non-essential gene) have been tested using this assay and intermediate values for precocious sister chromatid separation have been found (ranging from 15% to 30% compared to the 8% to 10% for w i l d type cells). The function o f many o f these proteins is unclear as they do not appear to be part o f the cohesin complex or part o f the chromatin loading process. M a n y o f the proteins have been categorized as factors needed for establishment o f cohesion during S phase, although with non-essential proteins, the exact time that they are needed is difficult to determine (Mayer et al., 2001; Mayer et al., 2004; Warren et al., 2004).  29  A n interesting subset o f the non-essential genes involved in cohesion appear to work together in an alternative R F C complex. C t f l 8 p replaces R f c l p i n the R F C complex and is found i n a complex with the other four subunits (Rfc2,3,4 and 5) as w e l l as CtfBp and D c c l p (Mayer et al., 2001). This alternative R F C complex plays a role i n the establishment o f sister chromatid cohesion and deletion mutants o f CTF8, CTF18 and DCC1, as well as mutants of RFC4 show cohesion defects when tested b y the precocious sister chromatid separation assay (Mayer et al., 2001). One model for the mechanism o f action o f this alternative R F C complex is that it is responsible for loading or unloading P C N A at replication forks in order to carry out a polymerase switch. The alternative R F C complex can load P C N A weakly onto D N A as well as unloading P C N A from D N A in vitro (Bylund and Burgers, 2005). This polymerase switch would facilitate D N A replication through sites o f cohesion containing the cohesin complex (Mayer et al., 2001). Initially this idea looked promising as a new D N A Polymerase, Polymerase a , was found and mutations i n its subunits (TRF4 and TRF5) displayed cohesion defects. Recently it has been shown however that polymerase a may be a poly(A) polymerase instead o f a D N A polymerase, and the pleitropic effects o f mutations may be an indirect consequence o f altered protein expression (Saitoh et al., 2002; Read et al., 2002). There are many intriguing connections between the replication fork machinery and sister chromatid cohesion. Temperature sensitive mutations in E c o l p / Ctf7p, required for establishing cohesion at S phase, can be suppressed by overexpression o f P C N A (Skibbens et al., 1999) and E c o l p / Ctf7p can associate with R F C components (Kenna and Skibbens, 2003). Cohesion establishment may also occur shortly after the replication fork has passed by using P C N A that has stayed bound to D N A . Other genes  30  that have been implicated i n sister chromatid cohesion include C h l l p , a D N A helicase, M r e l l p involved i n D N A damage checkpoint signaling (discussed below), Ctf4p, and T o f l p . T o f l p is a topoisomerase I interacting protein that is involved i n the D N A damage response pathway i n S phase. Proteins involved i n microtubule dynamics such as Kar3p and B i m l p also have cohesion defects and the mechanism for this defect is unclear (Mayer et al., 2004; Warren et al., 2004). Recent work has implicated the R S C (remodel the structure o f chromatin) nucleosome remodeling complex i n sister chromatid cohesion (Baetz et al., 2004; H s u et al., 2003; Huang et a l , 2004; Huang and Laurent, 2004). R S C is a multi-subunit complex that is part o f the S W I / S N F family o f ATP-dependent chromatin remodelers (Wang, 2003). Their main function is to reposition nucleosome arrays and change the underlying chromatin structure o f chromosomes i n order to facilitate or repress transcription. R S C contains distinct sub-complexes containing either R s c l p or Rsc2p and these sub-complexes have different effects on sister chromatid cohesion and chromosome segregation. rsc2A mutants have a high rate o f chromosome missegregation and more severe sister chromatid cohesion defects than rsclA mutants. There are conflicting reports o f whether or not the R S C complex is necessary for cohesin loading w i t h one group claiming that R S C mutants have defective loading o f S c c l p onto chromosome arms (with centromere loading unaffected) (Huang et al., 2004) while other groups have not seen this defect (Baetz et al., 2004). The R S C mutants do not appear to be involved in maintaining cohesion i n G 2 / M . Transcription o f genes involved i n sister chromatid cohesion is not down-regulated i n transcriptional profiling experiments performed on R S C mutants so the effect o f R S C on sister chromatid cohesion is likely not due to R S C  31  remodeling chromatin to allow access o f transcription factors to such genes. The R S C complex may play a role in the positioning o f nucleosomes during replication and i n that manner help to establish sister chromatid cohesion during replication.  Meiotic sister chromatid cohesion Sister chromatid cohesion and kinetochore dynamics must be modified during meiosis (Petronczki et al., 2003; Watanabe, 2004). Meiotic division couples two rounds o f chromosome segregation to one round o f D N A replication, thereby forming I n haploid progeny from a 2n diploid parent. In the first round o f meiosis, homologous chromosomes are paired, bivalents are formed and homologous chromosomes undergo recombination facilitated by the synaptonemal complex. Sister kinetochores must attach to the same spindle pole body, and this attachment is promoted by a meiosis specific kinetochore protein, monopolin, M a m l i n budding yeast (Toth et al., 2000). Rec8p, a variant o f S c c l p , replaces S c c l p in the cohesin complex during meiosis (Buonomo et al., 2000). Rec8p that is part o f cohesin on the chromosomal arms, but not Rec8p at the centromere, is cleaved by E s p l p during meiosis I (after P d s l p is ubiquitinated and degraded by the A P C , as i n mitosis), and Rec8p containing cohesin is released from chromosome arms leading to loss o f sister chromatid cohesion and the resolution o f chiasmata. Rec8p containing cohesin on chromosome arms provides the attachment between homologous chromosomes, and once dissolved, homologous chromosomes (each consisting o f two sister chromatids) are pulled to opposite spindle poles (Buonomo et al., 2000). Rec8p is protected from cleavage at the kinetochore b y S g o l p and this residual cohesin complex provides cohesion until the second round o f meiosis (Kitajima  32  et al., 2004; Marston et al., 2004; Katis et al., 2004). During the second round o f meiosis, Rec8p is cleaved at the centromere, Rec8p containing cohesin is released from the centromere, and centromeric cohesion is dissolved, allowing sister chromatids to separate (Klein et al., 1999; Buonomo et al., 2000). S g o l p was initially characterized as a protein important for meiosis and Rec8p function but recent work has shown that it is also necessary for proper mitotic chromosome segregation and is involved i n mitotic kinetochore and checkpoint functions sensing tension (Watanabe and Kitajima, 2005; Indjeian et al., 2005). S g o l p ' s roles both at meiosis and mitosis highlights the need to study both processes for a clearer understanding o f chromosome segregation.  Sister Chromatid Cohesion and DNA damage checkpoint and repair The detection of, response to, and repair o f D N A damage is o f fundamental importance to genome stability. Double strand breaks ( D S B ) are particularly dangerous as they cause a disruption to both strands o f D N A ( A y l o n and Kupiec, 2004). D S B s that are not dealt with correctly can lead to genome instability. Mechanisms have therefore evolved to respond to D N A damage that occurs spontaneously i n the cell cycle (e.g. replication fork stalling and collapsing) as well as from environmental insults (e.g. ionizing radiation). There are two major pathways for "the repair o f D S B s , nonhomologous end joining ( N H E J ) and homologous recombination ( H R ) ( A y l o n and Kupiec, 2004). H R does not result i n the loss o f genetic material (i.e. D N A sequences surrounding the D S B ) and is the primary pathway o f repair o f D S B s i n budding yeast (Friedl et al., 1998; Lisby et al., 2004). It requires the presence o f a template, either a sister chromatid or a homologous chromosome, i n order to repair the D S B and therefore  33  much o f this repair process occurs after replication when there is a sister chromatid as a template. Sister chromatid cohesion is important for this process and i n budding yeast it has been shown that cohesion established during D N A replication is necessary for proper repair o f D N A D S B s (Sjogren and Nasmyth, 2001). There has been some debate about whether the cohesion established during S phase, per se, is the requirement for postreplicative repair or i f there was some other direct function o f the cohesin complex in D S B repair. In human cells, cohesin has been shown to accumulate and localize at sites o f D S B s ( K i m et al., 2002) and a cohesin subunit ( S m c l ) is phosphorylated by A T M ( T e l l ) i n response to D N A damage induced b y ionizing radiation (Kitagawa et al., 2004; K i m et al., 2002b; Y a z d i et al., 2002). This phosphorylation is necessary for the checkpoint response. Recent work i n budding yeast has shown that cohesin does in fact localize to sites o f D N A D S B s and that this localized concentration o f cohesin is necessary for efficient repair o f D S B s . Through a series o f elegant experiments using the H O endonuclease (a site specific nuclease that w i l l generate a D S B ) and Chromatin Immunoprecipitation (ChIP) two groups were able to show that i n budding yeast the cohesin complex is recruited to flanking regions o f a D S B (up to 100 kbases, 50 kbases each side o f the D S B ) (Unal et al., 2004; Strom et al., 2004). This recruitment o f cohesin is required for efficient repair of D S B s and is dependent on Scc2p and Scc4p complex, the complex that loads cohesin onto chromatin. Cohesin that is recruited during the D N A damage response is functional and can prevent sister chromatid separation (Strom et al., 2004). This recruitment o f cohesin is also dependent on the phosphorylation o f Histone H 2 A X (the major Histone H 2 A variant i n budding yeast) at sites o f recruitment by M e c l p and T e l l p (Unal et al.,  34  2004). M e c l p and T e l l p are phosphoinositide 3-kinase-like kinases involved i n the D N A damage checkpoint response pathway and recognition o f D N A damage sensors (discussed below). Rad53p and M r e l l p were also shown to be necessary for cohesin recruitment to sites o f D S B . M r e l l p is i n a complex ( M r e l Ip/Rad50p/Xrs2p, M R X complex) that localizes to D S B s and may serve as the early sensor and indicator o f D N A damage and Rad53p is a kinase involved i n transducing the D N A damage checkpoint signal (see below). The likely function o f cohesin recruited to D S B s is to provide sister chromatid cohesion and ensure that the right sequences are used as donor templates i n H R repair (Sjogren and Nasmyth, 2001; Strom et al., 2004; U n a l et al., 2004). A r e there other functions that cohesin is playing at sites o f D S B s and D N A damage? In human cells the two S M C subunits o f cohesin have also been shown to be required for the S phase checkpoint signaling pathway and it is not clear i f this activity is independent o f their functions i n the cohesin complex (Lehmann, 2005). M a n y o f the deletion mutants o f the non-essential genes coding for factors involved i n sister chromatid cohesion are also hyper-sensitive to D N A damaging agents. CTF8, CTF18, CHL1, DCC1, and CTF4 deletion mutants are hyper-sensitive to D N A damaging agents such as ionizing radiation, bleomycin, and methylmethanesulfonate ( M M S ) (Game et al., 2003). While these factors are non-essential for viability i n budding yeast, their sensitivity to D N A damaging agents could represent their action i n dealing with D N A damage. O n the other hand, the sister chromatid cohesion defect may contribute to inefficient repair o f damage caused by D N A damaging agents and the hyper-sensitivity is simply the result o f the sister chromatid cohesion defect. Understanding the biochemical function o f non-essential genes and the time that they are  35  needed represents further challenges in understanding all the different aspects o f sister chromatid cohesion.  Checkpoints that Monitor Events and Damage during the Cell Cycle Checkpoints are mechanisms that ensure that events i n the cell cycle occur i n the proper order and that the cell cycle is proceeding with no defects that may cause problems (Hartwell and Weinert, 1989). Checkpoint mechanisms w i l l halt progress through the cell cycle to allow cells time to repair defects (Kastan and Bartek, 2004). There are irreversible events during the cell cycle and executing these events before the cell is ready can have detrimental effects on survival. Checkpoint mechanisms generally consist o f three levels or steps; first there must be a mechanism for sensing the damage or defect, secondly the checkpoint must transduce or mediate this signal usually through protein kinases that phosphorylate substrates, and finally the signal must trigger responses through effector proteins such as arresting the cell cycle until repair is completed and also mobilizing factors that w i l l repair the damage (Nyberg et al., 2002). The checkpoint mechanisms that w i l l be discussed next are important for chromosome segregation and genome stability and include the D N A damage / S phase checkpoint and the spindle assembly checkpoint. These pathways are w e l l conserved from budding yeast to humans and the medical importance o f many o f the proteins involved i n these checkpoint functions is demonstrated by the mutations i n genes encoding these proteins that have been found i n tumour cell lines.  36  DNA Damage Checkpoint / S phase checkpoint Damage to D N A can be caused by sources external to the cell such as ionizing radiation or sources that are the result o f cell processes, such as free oxygen radicals that arise from normal cellular metabolism. During S phase replication forks can also become stalled i f the nucleotide pool is disrupted or i f there is a physical impediment to the replication forks such as D N A adducts caused by alkylating agents. A stalled replication fork that collapses would cause a D S B that requires repair. The D N A damage / S phase checkpoint is responsible for monitoring D N A for damage and responding to the damage (for reviews see Nyberg et al., 2002; Longhese et al., 2003; Murakami and Nurse, 2000). The signals that trigger the D N A damage checkpoint have not been completely elucidated. Because there are many forms o f D N A damage it is generally assumed that processing o f the D N A damage into specific forms is a part o f the process o f detecting and signaling damaged D N A . Single stranded D N A and D S B s have both been postulated to be important structures that constitute the signal that the D N A damage checkpoint is responding to (Nyberg et al., 2002). The R p a heterotrimer that binds single stranded D N A and is important for D N A replication is also important for D N A damage detection and response (Longhese et al., 1996; Santocanale et al., 1995). A s well, an alternative R F C complex with Rad24p replacing R f c l p can load a P C N A - l i k e complex consisting o f R a d l 7 p / D d c l p / M e c 3 p onto sites o f D N A damage (Majka and Burgers, 2003). A l s o , the M R X complex localizes to D S B s and acts as a signal at D S B s ( D ' A m o u r s and Jackson, 2002). These proteins are all involved i n signaling D N A damage and are upstream o f proteins involved i n cell cycle arrest and D N A repair.  37  The transducers that serve to amplify the D N A damage signal and help to mediate a response consist o f kinases that phosphorylate substrates i n response to the initial D N A damage signal. The two chief mediators are phosphoinositide 3-kinase related kinases ( P I K K s ) , M e c l p ( A T R i n human cells) and T e l l p ( A T M in human cells) (Abraham, 2001) . Biochemical evidence has suggested that M e c l p is important for amplifying the signal from single stranded D N A while T e l l p is more important for D S B signaling (Nyberg et al., 2002). Downstream kinases that are phosphorylated b y M e c l p and T e l l p include C h k l p and Rad53p, both o f which are important for inducing cell cycle arrest (Longhese et al., 2003). D N A damage at various stages o f the cell cycle can cause a halt i n cell cycle progression until there has been time to repair the damage. G 1 - , S- and G2-arrest can all occur i n response to D N A damage. The G l arrest i n response to D N A damage i n budding yeast is weak and lasts for approximately an hour. The response is mediated by Rad53p phosphorylation o f Swi4p/6p transcription factors thereby inhibiting and delaying the production o f G l cyclins (Sidorova and Breeden, 1997). Budding yeast cells can proceed through this temporary block into S phase even with D S B s . The likely reason for this weaker G l D N A damage response is that homologous recombination from sister chromatid templates is the major repair mechanism i n budding yeast (Nyberg et al., 2002) . D N A damage that is encountered i n S phase, such as D N A adducts caused b y alkylating agents, result in stalled replication forks due to the physical impediment o f the adduct as both checkpoint proficient and deficient cells have stalled replication forks (Tercero and Diffley, 2001). The D N A damage checkpoint i n S phase (dependent on many o f the factors involved i n the replication fork, as well as Rad9p, M e c l p and  38  Rad53p) has three functions; it prevents firing o f late origins o f replication, it maintains the stalled replication fork so that D N A replication can proceed once damage is fixed, and it prevents entry into mitosis (Tercero and Diffley, 2001). G2 D N A damage in budding yeast also causes arrest o f cells, preventing entry into mitosis using the same machinery that is used by the spindle assembly checkpoint (discussed below). The checkpoint prevents the metaphase to anaphase transition by maintaining high levels o f the securin, P d s l p , and preventing the release o f E s p l p and the subsequent degradation and dissociation o f cohesins from sister chromatids (Cohen-Fix and Koshland, 1997; Tinker-Kulberg and Morgan, 1999). The homologous recombination machinery (the M R X complex, the Rad52p epistasis group) is active i n G 2 to repair D S B s that have been generated directly or indirectly from processing o f other types o f D N A damage (Lisby and Rothstein, 2005). A s well as arresting cells and allowing time for repair, effector responses can mediate transcriptional responses that are required for repair or adaptation to D N A damage. Genes that have been shown to be transcriptionally regulated by D N A damage include genes involved in D N A metabolism such as RAD2, RAD 18, RAD54, CDC9, RNR2 and RNR3.  (Gasch et al., 2001)  T h e Spindle A s s e m b l y C h e c k p o i n t The spindle assembly checkpoint monitors proper bi-polar attachment o f the mitotic spindle to the kinetochores o f sister chromatids. Spindle damage (for example, caused b y microtubule depolymerizing drugs such as nocodazole or benomyl) w i l l also trigger the spindle assembly checkpoint. The checkpoint w i l l arrest cells i n G 2 / M by preventing the metaphase to anaphase transition. M a n y o f the proteins involved i n the  39  spindle assembly checkpoint were first isolated i n budding yeast and subsequently found to be conserved in multicellular eukaryotes. In budding yeast, the majority o f the spindle assembly checkpoint genes are not essential and were isolated in random mutagenesis screens identifying mutants hypersensitive to microtubule depolymerizing drugs (for reviews see L e w and Burke, 2003; Skibbens and Hieter, 1998; A m o n , 1999). The signal that the spindle assembly checkpoint responds to has been proposed to be either lack o f tension exerted at kinetochores or the lack o f microtubule binding at the kinetochores (Lew and Burke, 2003). The difficulty in distinguishing between the two possibilities is that both processes are tied together and tension may regulate microtubule binding and unattached kinetochores are not under tension. For example, I p l l p may destabilize kinetochore microtubule attachments that lack tension and signal this lack o f tension. Alternatively, the transient unattached kinetochores that have been caused b y I p l l p activity may be the signal (Tanaka, 2005). Both processes are likely important as cellular localization o f spindle assembly checkpoint components has shown differences dependent on the status o f kinetochore microtubule attachments and tension exerted on kinetochores (Lew and Burke, 2003). The components that are involved i n the spindle assembly checkpoint have been extensively characterized i n budding yeast. A n intact kinetochore is considered to be necessary for the checkpoint response as it provides a platform to allow detection o f impaired kinetochore microtubule attachments (Gardner et al., 2001; G o h and Kilmartin, 1993) and mutations i n members o f the C B F 3 complex have an effect on the checkpoint because the kinetochore does not assemble properly. There is a direct connection between the C B F 3 complex and the spindle assembly checkpoint as S k p l p has been  40  found to associate with B u b l p , and the association is necessary for signaling kinetochore tension defects (Kitagawa et al., 2003). The N D C 8 0 complex also appears to play a role in checkpoint response. The kinetochore remains largely intact i n mutants o f the N D C c 8 0 complex and this complex may play a role i n recruiting and regulating the proteins involved i n the checkpoint (Janke et al., 2001; M c C l e l a n d et al., 2003). M a d l p , Mad2p, Mad3p, B u b l p (a protein kinase) and Bub3p, as well as M p s l p (involved also i n S P B duplication and a protein kinase) are proteins important for the spindle assembly checkpoint (Lew and Burke, 2003; A m o n , 1999). The prevailing hypothesis on how the checkpoint induces cell cycle arrest is that in the event o f kinetochore microtubule defects, M a d 2 p is exchanged from a M a d l p / M a d 2 p complex to a Cdc20p/Mad2p complex. Cdc20p is one o f the adaptors for the A P C / C ( A P C / C (Cdc20p) is responsible for the ubiquitination and degradation o f P d s l p securin) and Mad2p binding o f Cdc20p prevents this activity, thus maintaining high levels o f P d s l p and preventing the release o f E s p l p , thus preventing the degradation and dissociation o f cohesins from sister chromatids (Luo et al., 2000; Sironi et al., 2002; Y u , 2002).  Chromosome Transmission Fidelity Mutants and Characterizing Novel Genes Involved in Chromosome Segregation In order to isolate and characterize genes involved i n chromosome segregation, different screens have been performed i n the past by monitoring loss o f a chromosome marker on minichromosomes, chromosome fragments, or endogenous chromosomes. The strategy has been to use these screens as a primary screen to isolate mutants that have reduced chromosome transmission fidelity as the sole criterion and to subsequently  41  characterize the collection using secondary screens. The chromosome transmission fidelity (ctf) mutant collection takes advantage o f an artificial chromosome fragment that was engineered to contain the SUP 11 suppressor gene (Spencer et al., 1990). The chromosome fragment was introduced into yeast strains that contained the ade2 mutation. The ade2 mutation causes a red pigment to accumulate i n yeast colonies while the SUP 11 suppressor w i l l suppress this mutation and the yeast colony w i l l be white. This provides a visual assay for marker loss by observing the rate o f sectoring (red sectors i n a white yeast colony) and was the basis for isolating mutants that increased the rates o f sectoring (and b y extension, the rate o f loss and non-disjunction o f endogenous chromosomes) compared to w i l d type yeast cells. The initial set o f ctf mutants were isolated using ethylmethanesulfonate ( E M S ) mutagenesis and led to the identification o f mutants that were involved in different aspects o f chromosome segregation such as kinetochore function, sister chromatid cohesion, and D N A metabolism. Early work in the Hieter laboratory focused on finding the ctf mutants that were involved i n kinetochore function. To identify the relevant mutants, secondary assays were developed. One example o f a secondary assay used is the centromere transcriptional readthrough assay assessing kinetochore proteins binding to C E N i n ctf mutants.  It employed a reporter gene under the G A L promoter introduced near the C E N  sequence. C t f mutants defective for kinetochore function allow expression o f the reporter gene while other mutants or w i l d type strains did not allow expression (Doheny et al., 1993). Another example is Synthetic Dosage Lethality ( S D L ) whereby overexpressing a gene involved i n kinetochore function may have no effect in w i l d type cells but may cause lethality i n a kinetochore defective mutant ( K r o l l et al., 1996; H y l a n d et al., 1999).  42  The lethality is most likely due to compromising what few proper kinetochores have assembled b y further upsetting the stochiometric balance in the cell. U s i n g these secondary assays, several ctf genes have been identified and subsequently characterized as members o f the inner and central kinetochore (such as C t f l 3 p , C t f l 4 p (NdclOp), C t f l 9 p , C t O p , Chl4p) (Hyland et al., 1999; Measday et al., 2002; Pot et al., 2003). , ' A n alternative approach is to employ genetic interaction screens using mutations in genes known to be important components o f the segregation machinery as genetic entry points for identifying additional genes important for chromosome segregation. H i g h copy suppression occurs when having multiple copies o f a gene that is functionally related to a second gene, rescues a phenotype o f mutants in the second gene (typically temperature sensitivity). H i g h copy suppression screens have been used to identify interacting proteins as w e l l as proteins involved i n similar biochemical pathways. SKP1 (Suppressor o f Kinetochore Protein) was isolated as a high copy suppressor o f the temperature sensitive mutation ctfl3-30 and was subsequently characterized to be a member o f the C B F 3 complex (Connelly and Hieter, 1996) as well as being a subunit o f the S C F complex (Bai et al., 1996). SGT1 (Suppressor o f the G 2 allele o f SKP1) was found as a high copy suppressor o f skpl-4 and was subsequently characterized as a protein partner o f S k p l at both the kinetochore and the S C F complex (Kitagawa et al., 1999). Further characterization o f the budding yeast kinetochore and other components o f the chromosome segregation machinery could therefore be undertaken with SGT1 as the potential springboard for genetic screens that may isolate novel interactors and regulators.  43  O v e r v i e w a n d Scope of the Thesis When this work began there were relatively few components o f the budding yeast kinetochore identified and characterized. In the last five years there has been a large increase i n the number o f proteins that have been identified and placed at the kinetochore. M a n y o f the proteins that have been placed i n the central kinetochore have functions that are largely unclear. Finding regulators o f these proteins and the yeast kinetochore could shed new light on many o f these proteins. W o r k i n the yeast community i n the last few years has also elucidated much o f the mechanism o f sister chromatid cohesion and how it affects bi-orientation o f sister kinetochores and also how it affects post-replicative D N A repair. The goal o f this work was to further characterize components important for chromosome segregation, initially at the budding yeast kinetochore but also at sister chromatid cohesion as the work continued to progress. W e began this work using SGT1 as a starting point. A class o f temperature sensitive mutations i n SGT1 display defects i n kinetochore function and we were interested i n identifying high copy suppressors o f the temperature sensitivity. W e reasoned that this may identify novel components or regulators o f the yeast kinetochore. To identify potential protein partners o f S g t l p , we carried out a two-hybrid screen using SGT1 as the "bait" against the genome wide collection o f O R F s i n collaboration with Stan Fields. Phenotypic analysis o f deletion mutants corresponding to genes that were identified i n the two screens focused our interest on an uncharacterized O R F , Y D R 0 1 4 W . M u c h o f the work i n the second half o f Chapter 2 and i n the majority o f Chapter 3 is focused on characterizing the role o f  44  Y D R 0 1 4 W (RAD61) i n chromosome segregation and the function o f a Rad61p protein interaction partner, D e d l p .  45  Chapter 2:  Analysis oiSGTl genetic and physical interactions and identification of a factor involved in chromosome segregation, RAD61 / CTF6  46  Introduction T w o approaches were undertaken to identify genes that could be important for chromosome segregation and i n particular, kinetochore function. Both approaches attempted to identify proteins that were playing a role at the budding yeast kinetochore i n concert with S g t l p . The first approach utilized strains that contained the sgtl-3 mutation that causes arrest i n G 2 / M at non-permissive temperature. W e looked for genes that when present i n multiple copies, could suppress the temperature sensitive phenotype o f sgtl-3 mutant strains. This high copy suppression screen identified four O R F s , that when present i n multiple copies, rescued the lethality o f sgtl-3 strains at 37°C. The second approach was to identify protein partners o f S g t l p b y using SGT1 as the "bait" i n a genome wide two-hybrid screen i n collaboration with Stan Fields. W e focused on Y D R 0 1 4 W / RAD611 CTF6 because the deletion mutant displayed a chromosome missegregation phenotype. The experiments that we performed i n the second half o f the chapter were designed to characterize Rad61p's role i n chromosome segregation.  47  Materials and Methods Yeast strains, growth conditions and media Yeast strains used i n this study are listed i n Table 2-1. Strains were grown at 2 5 ° C unless otherwise indicated. Temperature sensitivity and lethality was assessed at 3 7 ° C . M e d i a for growth and sporulation were described previously (Rose et al., 1990). To visualize the loss o f the non-essential chromosome fragment (CF), the strains were first grown in S C media lacking either uracil or tryptophan (selecting for the C F ) , then either plated (200 to 300 cells per plate) or streaked to single colonies onto S C complete media with 65 fiM adenine concentration. Colonies were visualized after growth at 2 5 ° C or the temperature indicated for 3 to 4 days followed by 1 or 2 days at 4 ° C to increase the resolution o f the red versus white sectors. Cells that contain the C F w i l l appear as white colonies while cells that have lost the C F w i l l appear as red colonies. For assays testing sensitivity to microtubule depolymerizing drugs, benomyl from DuPont (Wilmington, D E ) was dissolved i n dimethylsulphoxide ( D M S O ) at 10 m g / m L and added to the indicated concentrations to Y P D plates, with addition o f D M S O lacking drug as a control. Methlymethanesulfonate ( M M S ) i n liquid form (99% pure, from Sigma) or bleomycin (10 Units/mL dissolved i n distilled water) was added to Y P D plates at the indicated concentrations. Epitope tagging and deletion o f genes were performed at the endogenous loci based on Longtine et al. (1998). Transformations o f yeast cells was performed according to Gietz and Schiestl (1995).  48  High Copy Suppressor Screen and Confirmation A 2u U R A genomic library (Connelly and Hieter, unpublished) was transformed into Y K K 66 strain carrying the sgtl-3 mutation, and transformants were first selected at 2 5 ° C for 3 days before replica plating to 37°C. W e identified - 3 0 0 yeast colonies that grew at 37°C from 4 x 10 yeast transformants and pooled them into 6 fractions o f 5  approximately 50 colonies each. W e purified plasmid D N A from the pooled colonies (containing plasmid D N A that rescued the conditional lethality at 37°C o f sgtl-3). Purified plasmid D N A was transformed into E. coli and the bacterial transformants were screened by colony hybridization for SGT1, SKP1 and CTF13 genomic sequences. W e found 18% o f the bacterial colonies contained an insert with SGT1, 1% SKP1, and 2 % CTF13.  O f the remaining transformants, we purified plasmid D N A from 70 colonies and  retransformed the plasmid D N A into Y K K 66 to confirm the suppression o f the temperature sensitivity phenotype. W e found four transformants that were able to suppress the conditional lethality o f sgtl-3 at 37°C. Sub-cloning using restriction sites present i n the genomic fragments was used to identify the O R F s that were responsible for suppression. Putative O R F s were cloned into G A L promoter containing vectors and transformed into Y K K 66. Y K K 66 strains containing the G A L vectors were allowed to grow on galactose or glucose plates at 25°C or 37°C to check that suppression was based on expression o f the O R F .  49  Co-immunoprecipitation experiments from yeast extracts Co-immunoprecipitations were performed as described previously i n Measday et al., (2002). In brief, yeast extracts were generated using glass bead lysis, equal amounts o f extracts (as measured by Bradford Assay) were incubated with conjugated A n t i - M Y C or A n t i - H A beads (Covance). Co-IPs were performed overnight at 4 ° C , washed i n extract buffer for a minimum o f 4 times, and eluted with sample buffer at 100°C for 4 minutes.  Genome wide two-hybrid assay SGT1 was cloned into p O B D 2 as described by Cagney et al., (2000). The pOBD2-S'G77 was functional based on complementation of sgtl-3 temperature sensitive mutants. The two-hybrid screen was performed as described b y Uetz et al., (2000). Putative two hybrid positives were retested and confirmed b y transforming the p O A D vectors containing the putative positives with pOBD2-S G77 and testing for interaction. ,  Fluorescence Activiated Cell Sorting (FACS) Analysis F A C S analysis was performed as previously described i n Hyland et al., (1999).  Chromosome Spreads and C h r o m a t i n Binding Assays Chromosome spreads were performed as previously described i n Michaelis et al., (1997). 9E10 A n t i - M Y C antibodies from Covance were used as the primary antibody with a C Y 3 - G A M secondary used to visualize R a d 6 1 - 1 3 M Y C p staining. D A P I was used as the marker for chromatin D N A . Chromatin Binding was performed as previously  50  described i n Donovan et al., (1997) with histone H 4 and Carboxypeptidase-Y used as controls for a chromatin binding protein and a cytoplasmic protein respectively.  Stability Assays on Rad61p The yeast mating pheromone, alpha-factor, was added to logarithmically growing cell cultures at concentrations o f 5 /ig/mL and strains were arrested for 3 hours at 25 °C. Cells were washed with Y P D and released into Y P D media. Protein extracts were made as previously described in Measday et al., (2002) and western blots performed using antiCdc28 antibodies and the 9E10 A n t i - M Y C antibody from Covance at the time points indicated as w e l l as preparing F A C S samples. For assays investigating the half life o f Rad61p, we added cyclohexamide to a concentration o f 10 pg/mL and protein extracts were made at the time points indicated. For testing the stability o f Rad61p i n sgtl-5 mutants compared to w i l d type, we cloned H A - R a d 6 1 into a G A L expression vector and transformed it into Y K K 57 (sgtl-5) and w i l d type strains. Cultures were grown i n liquid media selecting for the plasmid vector with glucose as the carbon source. Cells were subsequently harvested, washed and allowed to grow i n raffinose for 2 hours. Galactose was added to a concentration o f 2 % for 2 hours to induce HA-RAD61 transcription, time point 0 was upon subsequent addition o f glucose to repress transcription  oiHA-RAD61.  Microscopy Experiments and Indirect Immunofluorescence 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 autofluorescence.  Unless otherwise indicated microscope pictures are o f live cells harvested  51  during log growth, and resuspended i n an equal mixture o f F P M and 1.2% low melting point agarose to immobilize cells on the microscope slide. Cells were fixed b y addition o f 4% paraformaldehyde to equal amounts o f liquid culture (to a final concentration o f 2 % paraformaldehyde) for 10 minutes at room temperature and washed with S K (1 M Sorbitol, 50 m M K P C ^ p H 7.5) media. Microscope images were taken with a Zeiss A x i o p l a n microscope equipped with a C o o l S N A P H Q camera (Photometries) and Metamorph (Universal Imaging). Stacks or one focal plane o f images were taken as indicated i n the figure legends. Indirect immunofluorescence was performed as previously described i n Hyland et al., (1999). A n t i - M Y C (9E10) antibodies were applied at a dilution o f 1/1000 for 1 hour at 37°C, and secondary fluorescent antibody G A N - F or G A N - R was applied at a dilution o f 1/1000 at 37°C for 1 hour also. D A P I was contained in the mounting media.  G A L - H O experiments and Double Strand Break induction experiments Plasmids containing the H O endonuclease under the control o f the G A L promoter (plasmid 132) were transformed into w i l d type, rad52A, and rad61 A haploid strains and lethality was investigated on Y E P plates with galactose as the carbon source instead o f glucose ( Y P G plates). Experiments involving the induction o f the H O endonuclease from the G A L - H O plasmid i n order to generate a double strand break and investigation o f the co-localization o f Rad52p to that double strand break site were performed as previously described i n L i s b y et al., (2003).  52  Table 2-1 List of yeast strains used in this chapter Strain  Genotype  YPH499  M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al  trplA63  YPH500  Mztaura3-52  trplA63  YPH501  M a t a / a ura3-52/ura3-52 Iys2-801/lys2-801 ade2-101/ade2101 his3A200/his3A200 leu2Al/leu2Al trplA63/trplA63 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 sog2A::HIS3 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 sog2A::HIS3 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 sog2A::HIS3 CFIII (CEN3.L.) TRP1 SUP11 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 sog3A::HIS3 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 sog3A::HIS3 CFIII (CEN3.L) TRP1 SUP11 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63  YBC15 YBC16 YBC17 YKK560 YKK556 YKK529 YBC108 YBC109 YKK57 YKK190 YCTF7 YCTF40 YCTF60 YCTF134 YPH311  Reference  lys2-801 ade2-101 his3A200 leu2Al  sgtlA::HIS3 6HA-SGT1-LEU2 @ Ch.ni M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al SOG2-13MYC-TRP1 sgtlA::HIS3 6HA-SGT1-LEU2 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al SOG3-13MYC-TRP1 sgtlA::HIS3 6HA-SGT1-LEU2 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al sgtl-5:LEU2 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al sgtl-3:LEU2 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al CFIII (CEN3.L.) URA3 SUP 11 ctf6-7 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al CFIII (CEN3.L.) URA3 SUP 11 ctf6-40 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al CFIII (CEN3.L.) URA3 SUP 11 ctf6-60 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al CFIII (CEN3.L.) URA3 SUP 11 ctf6-134 M a t a ade2-101 his3A200 ura3-52 tubl-1  53  K . Kitagawa K . Kitagawa K . Kitagawa  This study K . Kitagawa  trplA63 trplA63 trplA63 trplA63 trplA63  ura3-52 tub2-104  M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al RAD61-13MYC-HIS3 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al RAD61-13MYC-HIS3  This study  K . Kitagawa  YBC197 YBC198  This study  This study trplA63 @ Ch.III trplA63 @ Ch.III trplA63  YPH312 M a t a ade2-l01  Sikorski and Hieter., 1989 Sikorski and Hieter., 1989 Sikorski and Hieter., 1989 This study  Spencer et al., 1990 Spencer et al., 1990 Spencer et al., 1990 Spencer et al., 1990 Spencer et al., 1990 Spencer et al., 1990 This Study  trplA63 This study trplA63  Table 2-1 List of yeast strains used in this chapter, continued YBC204 YBC205 YBC207  YBC309  YBC200 YBC201 YPH1555 YBC671 YBC672 YBC673 YBC674 YBC658  YBC661  YBC675 YPH1474 YPH1020  This study M a t a uraS-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 CFIII (CEN3.L) TRP1 SUP 11 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 CFIII (CEN3.L) TRP1 SUP11 Mata/aura3-52/ura3-52 Iys2-801/lys2-801 ade2-101/ade2-101 his3A200/his3A200 leu2Al/leu2Al trplA63/trplA63 rad61A::HIS3/rad61A::HIS3 M a t a / a ura3-52/ura3-52 Iys2-801/lys2-801 ade2-101/ade2-101 his3A200/his3A200 leu2Al/leu2Al trplA63/trplA63 rad61A::HIS3/rad61A::HIS3mad2A::HIS3/mad2A::HIS3 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 sgtlA::HIS3 6HA-SGT1-LEU2 @ Ch.IIIRAD61 -13MYC-TRP1 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 sgtlA::HIS3 6HA-SGT1-LEU2 @ Ch.III RAD61-13MYC-TRP1 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 NDC10-13MYC-kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 RAD61-VFP-kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 RAD61-VFP-kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 RAD61-VFP-kanMX6 NIC96-CFP-hphMX M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 RAD61-VFP-kanMX6 NIC96-CFP-hphMX M a t a ade2-l barl::LEU2 trpl-1 LYS2 RAD5 RAD52-CFP ura3::3xURA3-tetOxll21-Scel(ura3-1) his3-l 1,15::YFP-Laclhis3-x leu2-3,112::LacO-LEU2 HO-iYCL018W(leu2-3,l 12) TetR-RFP(iYGL119W) (W303 background) M a t a ade2-l barl::LEU2 trpl-1 LYS2 RAD5 RAD52-CFP ura3::3xURA3-tetOxll21-Scel(ura3-1) his3-ll,15::YFP-Ladhis3-x leu2-3,112::LacO-LEU2 HO-iYCL018W(leu2-3,l 12) TetR-RFP(iYGL119W) rad61A::HIS3 (W303 background) M a t a his3Al leu2A0 metl5A0 ura3A0 rad52A::kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 CTF8-13MYC-HIS3 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 CFIII (CEN3.L) TRP1 SUP 11  54  This study This study  This study  This study This study Pot el al., 2003 This study This study This study This study L i s b y et al., 2003  This study  Giaever et al., 2002 M a y e r et al., 2001 Spencer et al., 1990  Results Identification  of High Copy Suppressors of sgtl-3 (Suppressors of the G2 allele of  SGT1, SOGs) S g t l p is a protein that, together with its direct binding partner S k p l p , is essential for two distinct functions: 1) i n the assembly o f the budding yeast kinetochore, 2) and as a subunit o f the S C F E3 ubiquitin ligase complex (Kitagawa et al., 1999). SGT1 is an essential gene and, consistent with the two distinct functions o f the S k p l p / S g t l p complex, temperature sensitive mutants carrying mutations i n SGT1 fall into either o f two classes; at the restrictive temperature one class arrests at G l / S with small budded cells (sgtl-5), while the other class arrests at G 2 / M with large budded cells (sgtl-3) (Kitagawa et al., 1999). The G l / S alleles are defective i n S C F function while the G 2 / M alleles are defective in kinetochore function. H i g h copy suppressor screens led to the initial identification o f SKP1 as a suppressor of ctf 13-30, and SGT1 as a suppressor o f skpl-4 (Connelly and Hieter, 1996; Kitagawa et al., 1999). In an attempt to identify additional novel genes important for budding yeast kinetochore function, we performed a multicopy suppressor screen o f an sgtl-3 mutant. A genomic library carried i n a high copy plasmid was transformed into a yeast strain that harbored the sgtl-3 mutation and a screen was performed for genomic segments that could rescue the lethality o f the strain when grown at 37°C (see Materials and Methods for the details o f the screen). Genomic fragments that could suppress the temperature sensitivity o f sgtl-3 were sub-cloned to find the minimal sequences that could still suppress the temperature sensitivity. Four Open Reading Frames (ORFs) that were not SGT1, SKP1, or CTF 13 were identified that could suppress the temperature  55  sensitivity o f sgtl-3.  The four O R P s identified were Y L R 3 8 8 W (RPS29A), Y L R 0 7 3 C ,  Y D R 2 6 6 C , and Y D L 1 9 0 C (UFD2).  W e provisionally named Y L R 0 7 3 C and Y D R 2 6 6 C  SOG2 and SOG3 for Suppressors o f the G 2 allele o f SGT1. To confirm that expression o f these O R F s could indeed suppress the temperature sensitivity phenotype o f sgtl-3, we cloned each O R F into a vector that contained a galactose inducible promoter and showed that i n each case the suppression o f the temperature sensitivity depended on the expression o f the O R F s . A n example suppression o f the temperature sensitivity phenotype o f sgtl-3 b y SOG2 or RPS29A,  as well as a negative control, Y L R 3 8 7 C , and  vector alone, is shown i n Figure 2-1. Expression o f Sog2p and Rps29ap i n sgtl-3 strains rescued the temperature sensitivity phenotype o f the sgtl-3 strain and allowed growth at 3 5 ° C whereas vector alone or expressing the protein product o f Y L R 3 8 7 C did not rescue the temperature sensitivity phenotype and the strains did not grow at 35°C.  SOG2 and SOG3 do not encode Sgtlp binding proteins and are not required for chromosome segregation RPS29A is a ribosomal structural subunit (91% identical to RPS29B i n budding yeast and 67% identical to human RPS29) and its function is well characterized i n m R N A binding and translation. UFD2 is a gene involved i n assembly o f multiubiquitin chains on ubiquitin-protein conjugates. The deletion mutants o f RPS29A and UFD2 are viable and do not have chromosome missegregation phenotypes as measured by three different screens, the sectoring assay (loss o f a non-essential chromosome fragment), the a - like faker mating screen, and the bi-mater screen (Karen Yuen, personal communication) and,  56  GAL-S0G2 YKK190  GAL vector YKK190  GAL-YLR387C YKK190  GAL-YLR387C YKK190  YKK190 GAL-YLR388W (RPS29A) 25°C Galactose galactose  35°C galactose  Figure 2-1 - Suppression of the temperature sensitivity of sgtl-3 by overexpression of SOG2 and RPS29A. A n example o f suppression. Vectors carrying the SOG2 ( Y L R 0 7 3 C ) , RPS29A ( Y L R 3 8 8 W ) , and a candidate gene ( Y L R 3 8 7 C - negative) with a Galactose inducible promoter, were transformed into strains carrying the temperature sensitive allele sgtl-3. Strains were then streaked and allowed to grow at 25°C or 35°C for 4 days on galactose media. SOG2 and RPS29A when overexpressed were able to suppress the lethality o f sgtl-3, but not the vector alone or Y L R 3 8 7 C .  57  therefore, these two genes were not further characterized. SOG2 and SOG3 were previously uncharacterized O R F s that coded for proteins o f unknown function. W e performed phenotypic assays on the deletion mutants o f SOG2 and SOG3 i n order to investigate any potential role o f Sog2p or Sog3p i n chromosome segregation. Neither sog2A nor sog3A strains exhibited increased rates o f missegregation o f a marker chromosome fragment relative to w i l d type (data not shown). The strains also did not show sensitivity to the microtubule depolymerizing drug benomyl (Figure 2-2). B e n o m y l sensitivity is a hallmark o f genes that are involved i n kinetochore or sister chromatid cohesion function. In addition, SOG2 and SOG3 did not display genetic interactions such as S D L with components o f the inner and central kinetochore. For example, when we overexpressed CTF13, NDC10, SKP1, SGT1, and CTF19 from galactose inducible promoters i n sog2A and sog3A strains, there was no effect on growth o f the deletion mutants, as compared to w i l d type strains (data not shown). W e performed co-immunoprecipitation experiments using yeast extracts to determine i f either Sog2p or Sog3p interacted physically with S g t l p . Epitope tagged constructs o f Sog2p, Sog3p and S g t l p were made at their endogenous loci. Yeast extracts were prepared from strains containing SOG2-13MYC in Figure 2-3a and 2-3b), SOG3-13MYC  and SGT1-HA (lanes 1 to 3  and SGT1-6HA (lanes 4 to 6 i n Figure 2-3a and  2-3b), and SGT1-6HA alone as a negative control (lanes 7 to 9 i n Figure 2-3a and 2-3b). A n a n t i - M Y C IP was performed on the extracts and we did not detect any interaction between S g t l - 6 H A p and S o g 2 - 1 3 M Y C p or S o g 3 - 1 3 M Y C p (Figure 2-3b, see lanes 3 and 6). F r o m this co-immunoprecipitation experiment it appears that neither Sog2p nor Sog3p physically interact with S g t l p .  58  sog2A sog3A  Wild Type tub1-1 tub2-104  •• •• ••  YBC17 •ft YKK560 YPH499 * ft YPH311 YPH312  • • ft # *  DMSO  ft>  #• •  10/yg/mL benomyl  tub1-1 tub2-104  YBC17 YKK560 YPH499 YPH311 YPH312  Wihd Type  ^ ^ ^ ^ ^ ^ ^  YPH499  15/vg/mL benomyl  sog2A sog3A  Wild Type  [ 1 1 1  B  Figure 2-2 - sog2 and sogi deletion mutants show similar sensitivity to benomyl as wild type strains. Serial dilutions (lOx) were plated on media containing increasing amounts o f benomyl. tubl-1 (sensitive to benomyl) and tub2 mutants (resistant to benomyl) were used as controls. Cells were grown at 25°C for 5 days.  B y phenotypic analysis o f the deletion mutants and co-immunoprecipitation tests for protein-protein interaction i n whole cell extracts, we determined that Sog2p and Sog3p were not important for chromosome segregation and do not interact with S g t l p . W e therefore did not pursue further analysis o f the suppressor genes.  59  Figure 3 Sog2p and Sog3p do not co-IP with Sgtlp in yeast extracts a) A n t i - M Y C IP, A n t i - M Y C Western  Figure 2-3 - Sog2p and Sog3p do not co-IP with Sgtlp in yeast extracts.  YBC108 YBC109 YKK529 SOG2-13MYC SOG3-13MYC SGT1-6HA SGT1-6HA SGT1-6HA tot sup IP tot sup IP tot sup IP  Yeast extracts from strains expressing S O G 2 - 1 3 M Y C p and Sog3-i3MYC  1 2 3  4 5  P  6 7 8 9  b)Anti-HA Western YBC108  YBC109  YKK529  SOG2-13MYC SOG3-13MYC SGT1-6HA SGT1-6HA SGT1-6HA tot sup IP tot sup IP tot sup IP  Sgti-6HAp 1 2 3  4  5  6  S G T l - 6 H A p (lanes 1 to 3),  S O G 3 - 1 3 M Y C p and S G T 1 6Hap (lanes 4 to 6), or S G T 1 6Hap (lanes 7 to 9) were used for immunoprecipitation using A n t i - M Y C beads (9E10). A ) A n t i - M Y C (9E10) Western to show the IP o f the M Y C tagged constructs, we loaded an excess amount o f IP sample to detect any interaction thus the large amounts seen i n the IP lanes (3 and 6). B ) A n t i - H A (12CA5) W  e  g  t  e  m  t Q  d e t e c t  S  g  t  l  6  H  a  p  m  the IPs. S g t l - 6 H A p appears as a doublet. Lanes 7 to 9 were used as a negative control for non-specific binding o f S g t l 6 H A p to the A n t i - M Y C beads.  7 8 9  Identification of an ORF, YDR014W, as an SGT1 two hybrid interacting gene required for chromosome transmission fidelity A s an alternative approach to identifying novel genes involved i n chromosome transmission fidelity, we performed a genome wide two hybrid screen using SGT1 as the protein partner "bait". SGT1 was cloned into a D N A binding domain vector (pOBD2) that created a fusion protein containing the G A L 4 D N A binding domain fused to SGTJ. The construct was then screened against the entire set o f yeast open reading frames fused to the G A L 4 activating domain. The two-hybrid screen identified several expected  60  interactors, including SKP1 and several F-box containing genes (CDC4, GRR1, and MET30) that are also members o f the S C F complex (Table 2-2). Y D R 0 1 4 W , an O R F o f unknown function, was also identified. When the Y D R 0 1 4 W gene was deleted, the deletion mutant missegregated an artificial marker chromosome fragment at a rate higher than w i l d type (Figure 2-4). W e performed a half-sector analysis by counting colonies  Table 2-2 Genome wide two hybrid interactions with SGT1 Bait  Target  ORF  Function  SGT1  SKP1  YDR328 C  Kinetochore complex and component of SCF  CDC4  YFL009 W  F-box protein and component of SCF complex  GRR1  YJR090 C  F-box protein and component of SCF complex, glucose repression and glucose and cation transport  MET30  YIL046 W  F-box protein and component of SCF complex, regulates sulfur assimilation genes  YLR368 W  YLR368 W  F-box protein, unknown function  RPN7  YPR108 W  Proteasome  BDF1  YLR399 C  Required for sporulation, has two bromodomain motifs and one ET domain  YMR111 C  YMR111 C  Unknown  RAD61  YDR014 W  Unknown  61  Figure 2-4 - rad61A mutants sector. Haploid rad61A cells were plated onto Synthetic Complete media containing 65 p M adenine and compared to w i l d type strains. B o t h strains contain a Chromosome Fragment III that is marked with SUP 11 and TRP1. Red indicates loss o f chromosome fragment and white indicates retention o f the chromosome fragment.  from w i l d type or rad61A strains that contained half-sectors (or more) o f red pigment in order to quantify the chromosome missegregation rate o f rad61A strains and found that rad61A strains had a ~ 20x elevated rate o f chromosome missegregation compared to the w i l d type (data not shown). To test i f the two-hybrid interaction identified a protein-protein interaction in yeast cells we performed a co-immunoprecipitation experiment on yeast extracts expressing epitope tagged S g t l p and Rad61p ( Y D R 0 1 4 W , see below). W e used strains expressing only SGT1-6HA (lanes 1 to 3 in Figure 2-5a, and lanes 1 and 2 i n Figure 2-5b) or RAD61-13MYC  (lanes 4 to 6 in Figure 2-5a, and lanes 3 and 4 i n Figure 2-5b) as  negative controls and we used two strains expressing SGT1-6HA and  RAD61-13MYC  (lanes 7 to 12 in Figure 2-5a, and lanes 5 to 8 in Figure 2-5b) to test for interaction. W e could not detect an interaction between the two proteins (see lanes 6 and 8 in Figure 25b) implying that the interaction may be transient and sub-stochiometric, or that the initial two-hybrid result was a false positive.  62  a) Anti-MYC IP, Anti-MYC Western YKK529 YBC197 Sgt6 HA  Rad6113MYC  T SIP T  S IP  YBC200 YBC201 Rad61-13MYC Sgt1-6HA #1 #2 T  S IP  T  S  IP  Rad61-13MYCp  14 5 6 7 8 9  10 11 12  b) Anti-HA Western YKK529 YBC197 Sgt16HA  Rad6113MYC  YBC200  YBC201  Rad61-13MYC Sgt1-6HA #1 #2  Tot IP Tot IP Tot IP Tot IP  1  2  3  4  5  6  7  8  Figure 2-5 - Co-immunoprecipitation from yeast extracts does not indicate an interaction between Rad61-13MYCp and Sgtl-6HAp. A ) Yeast extract from strains expressing S g t l - 6 H A p (lanes 1 to 3), Rad611 3 M Y C p (lanes 4 to 6), and two strains expressing S g t l - 6 H A p and R a d 6 1 - 1 3 M Y C p (lanes 7 to 12) were prepared and A n t i - M Y C IPs performed. The single tagged strains were used as negative controls. B ) Samples from Figure 5a were rerun (only total yeast extracts and EP samples). Lanes 1 to 4 are the single tagged control strains, total and IP samples, lanes 5 to 8 are the two strains co-expressing S g t l - 6 H A p and R a d 6 1 - 1 3 M Y C p , total and IP samples.  63  YDR014W corresponds to a ctf mutant (CTF6) as well as a rad mutant (RAD61) In a parallel study, Y D R 0 1 4 W was isolated as an O R F able to complement the sectoring phenotype caused by mutant alleles within the CTF6 complementation group (Karen Y u e n , personal communication). To determine whether Y D R 0 1 4 W was indeed CTF6, the entire Y D R 0 1 4 W O R F was sequenced for each o f four independent isolates within the CTF6 complementation group. Single point mutations were identified in the coding region o f Y D R 0 1 4 W i n each case. The mutations were nonsense mutations that produced truncated protein products that were therefore likely non-functional (Table 2-3). The mutations fell into two classes; ctf6-7 and ctf6-60 were identical and ctf6-40 and ctf6134 were identical, suggesting that the pairs o f alleles were "siblings" derived from outgrowth o f the mutagenized cells in the original mutagenesis protocol that generated the ctf collection. In a diploid mating test assay, ctf6-40 / ctf6-134 (homozygous G 1 2 2 7 A , T R P to S T O P ) mutant diploids exhibited chromosome III missegregation at a rate lOOx higher than w i l d type strains (Spencer et al., 1990). W e conclude that Y D R 0 1 4 W encodes the gene that corresponds to the CTF6 complementation group and is important for chromosome segregation.  Table 2-3 RAD61 mutant alleles in the CTF mutant collection RAD61 allele  DNA Mutation  Amino Acid  ctf6-7  Base pair 1226 G to A  409 TRP to STOP  ctf6-40  Base pair 1227 G to A  409 TRP to STOP  ctf6-60  Base pair 1226 G to A  409 TRP to STOP  ctf6-134  Base pair 1227 G to A  409 TRP to STOP  ctf6-53  Not Sequenced  Not Sequenced  1 Mutations that were found were seen in a minimum of two sequencing reactions  64  Y D R 0 1 4 W was also independently identified as a deletion mutant that is sensitive to ionizing radiation i n a screen performed on the yeast genome wide non-essential gene deletion set and named RAD61 (Bennett et al., 2001; Game et al, 2003). rad6lA  strains  are sensitive to bleomycin (a gamma radiation mimetic causing double strand breaks) as well as M M S (a methylating agent), phenotypes that we were able to confirm i n our laboratory strain background (Figure 2-6a). To be consistent with published nomenclature, Y D R 0 1 4 W w i l l be referred to as RAD61.  rad61A  mutants exhibit a cell cycle delay in G2/M that is dependent on the spindle  assembly checkpoint rad61A haploid mutants displayed a sectoring phenotype indicating a much higher rate o f chromosome loss than wild type strains, but there were no growth effects at various temperatures tested (16°C to 37°C) (data not shown.) The haploid deletion mutants had approximately equal populations o f G l and G 2 cells i n logarithmically growing cell populations as measured b y F A C S , similar to w i l d type strains, and did not display increased sensitivity to benomyl, a microtubule depolymerizing drug (data not shown). However, rad61A /' rad61A homozygous diploid deletion mutants displayed a G 2 accumulation (Figure 2-6b, left hand panels). When we investigated cellular morphology o f rad61A /rad61A  compared to w i l d type diploids, we found a higher percentage o f cells  in a logarithmically growing population as short spindle pre-anaphase cells (29% i n rad61A / rad61A versus 13% i n w i l d type diploid cells). The G 2 accumulation was  65  dependent on Mad2p (Figure 2-6b, right hand panels). W e also found that rad61A / rad61A strains were resistant to intermediate levels o f benomyl, compared to w i l d type diploids (Figure 2-6c). Mad2p is a member o f the spindle assembly checkpoint that monitors and assures that sister kinetochores achieve bi-polar attachment to the spindle before allowing the metaphase to anaphase transition to occur. It has previously been shown that strains that have a defective kinetochore or a defect in sister chromatid cohesion may display a  >  G1 G2  b  G1  G2  Wild type I YPD  md61A Wild type  2.5 mU/mL bleomycin  5.0 mU/mL bleomycin  • •• •* YPD  0.01% MMS  0.03% MMS  Figure 2-6 - rad61A/ rad61A diploid cells have a G2/M accumulation dependent on Mad2p and are resistant to benomyl. A ) lOx serial dilutions o f rad61A ( Y B C 2 0 4 ) and w i l d type ( Y P H 4 9 9 ) cells were plated onto medium that contains bleomycin or M M S and grown for 5 days at 25°C. B ) F A C S analysis o f logarithmically growing w i l d type diploid ( Y P H 5 0 1 ) , rad61A/rad61A ( Y B C 2 0 7 ) , and rad61A mad2A/rad61A mad2A ( Y B C 3 0 9 ) strains. G l and G 2 peaks are labeled. C ) lOx serial dilutions o f w i l d type diploid (YPH501), rad61A/rad61A ( Y B C 2 0 7 ) , tubl-1 (YPH311), and tub2 mutant ( Y P H 3 1 2 ) cells were plated onto medium containing 0 to 15 ug/mL o f benomyl (in D M S O ) and grown for 5 days at 25°C.  66  Wild type diploid  rad61A/rad61A  -UJ  Lai  rad61b/rad61A  rad61Lmad2L/ rad61& madlL  (sensitive)  tub1-1 tub2-104 (m.)  0 ug/mL benomyl  Wild Type Diploid rad61A/rad61&  (sensitive)  tub1-1 lub2-104 Ires.)  c ,:•  Wild Type Diploid rad61&/rad61A  (sensitive) (res.) Wild Type Diploid tub1-1 Iub2-104  5 ug/mL benomyl  -0  OO ©  10 ug/mL benomyl  rad6U/rad61A  (sensitive) (res.) Wild Type Diploid tub1-1 tub2-W4  rad61A /rad61A  15ug/mL benomyl  G 2 accumulation that is dependent on the spindle assembly checkpoint (Hyland et al., 1999; Mayer et al., 2001); Rad61p could potentially function i n one o f these two processes. Strains with defective kinetochores or sister chromatid cohesion may also show benomyl sensitivity presumably due to the synergistic effects o f having perturbed microtubules and a defect i n kinetochore or cohesion function. The resistance o f the homozygous diploid deletion mutant to the drug benomyl is harder to explain and may be due to the slower progression through G 2 / M allowing cells more time to deal with depolymerized microtubules.  Rad61p is a stable nuclear protein RAD61 was identified as having a two-hybrid interaction with SGT1, but this interaction was not confirmed by a co-immuhoprecipitation experiment performed with yeast extracts. A possible explanation for this discrepancy could be that Rad61p is a substrate o f the S C F complex and the interaction is transient. To address this we investigated the stability o f Rad61p by quantifying the levels o f Rad61p at various stages in the cell cycle. Previous experiments b y others indicated that RAD61 m R N A levels are stable through the cell cycle (Saccharomyces Genome Database, S G D ) . W e synchronized yeast cell cultures with the addition o f a-factor mating pheromone (see Materials and Methods) causing a G l arrest. W e then released the cells from G l arrest by washing and removing the mating pheromone. Consistent with the R N A results, Rad61p protein levels were also constant throughout the cell cycle, (Figure 2-7a) as determined b y analysis o f yeast protein extracts by western blot using antibodies to an epitope tagged Rad61 protein and the F A C S analysis indicating cell cycle progression.  67  Anti-Cdc28p antibodies were used as a control for protein loading. The R a d 6 1 - 1 3 M Y C p fusion was functional as determined by complementation o f a rad61A mutation using the sectoring assay (data not shown). The constant level o f the Rad61 protein could be due to a long half life o f the protein or to constant expression o f Rad61p with a short half life. W e investigated the two possibilities by treating cells with cyclohexamide (a translation inhibitor). After treatment with cyclohexamide we prepared yeast extracts every 15 minutes. W e found that R a d 6 1 - 1 3 M Y C p appeared stable and had a half life much greater than that o f Clb2p, a mitotic cyclin targeted for degradation b y the A P C / C (Anaphase Promoting Complex) (Figure 2-7b) with a high turnover rate. W e concluded that Rad61p is a stable protein with a long half life. W e also tested to see i f overexpressed Rad61p could be a substrate for the S C F complex. W e cloned an H A - R a d 6 1 fusion protein into a vector with a galactose inducible promoter, and transformed the G a l - H A - R A D 6 1 vector into w i l d type and sgtl5 strains.  U p o n induction with galactose and subsequent repression o f expression with  glucose, we did not see stabilization o f H A - R a d 6 1 p i n the S C F mutant strains (Figure 27c) compared to w i l d type indicating that turnover o f excess Rad61p was not dependent on the S C F complex. W e conclude that Rad61p is not a substrate o f the S C F complex and its expression and levels are constant through the cell cycle. The phenotypic studies on rad61A mutants suggested that Rad61p could potentially function at the budding yeast kinetochore or in sister chromatid cohesion. To further investigate these possibilities, the cellular localization o f Rad61p was determined by indirect immunofluorescence microscopy using antibodies to epitope tagged Rad61p  68  ( R a d 6 1 - 1 3 M Y C p ) . In logarithmically growing cell cultures that were then fixed, Rad611 3 M Y C p showed a nuclear localization as determined by colocalization with D A P I stained D N A in all the cells examined (-150 cells in various stages o f the cell cycle) (Figure 2-8a). L i v e cell imaging o f logarithmically growing cell cultures was also undertaken using Rad61p tagged at the C-terminus with V F P (a G F P variant, Venus Fluorescent Protein) and integrated at the endogenous locus. The localization o f  a)  alpha-factor arrest and release I O Q CO  G 1  CO CJl —J CO —k —k —k O O O O - k O O C J l  o o o  RAD61-13MYCp|  G  2  [jjj  LOO  G1 G 2  IE  1 0 9  a - F 90  a-factor  Cdc28p  a - F 30  nz  i °- ° Mi  a - F 50  a - F 70  a - F 150  b) C y c l o h e x a m i d e treatment of log  c)  cultures  Wild type R  sgt1-5  G 30 60 90 R G 30 60 90  Minutes after cyclohexamide treatment 0  HA-RAD61p  15 30 45 60 75 -  CDC28p  69  Figure 2-7 - Rad61-13MYCp is stable through the cell cycle with a long half-life. A ) C e l l cultures from Y B C 1 9 8 were arrested i n alpha-factor for 2 hours at 3 0 ° C , washed, and released into Y P D at 30°C. F A C S and protein samples were taken at the time points indicated. Protein sample concentrations were determined using O D 2 8 0 readings, and Cdc28p was used as a loading control. B ) 10 />ig/mL o f cyclohexamide was added ( Y B C 1 9 8 ) to log phase cells (OD600 o f 0.6) and protein samples were extracted at the time points indicated. Protein sample concentrations were assessed with O D 2 8 0 readings and equal amounts loaded, with Cdc28p used as a loading control. C ) A G A L - H A R A D 6 1 plasmid was transformed into w i l d type cells (YPH499) and sgtl-5 ( Y K K 5 7 ) cells. L i q u i d cultures were grown i n selective media to maintain the plasmid overnight. Equal amounts o f cells (by absorbance readings at OD600) were resuspended i n rich media with glucose replaced by raffinose and grown for 2 hours. Galactose was than added to 2%, cells grew for another 2 hours and than glucose was added to repress transcription o f H A - R A D 6 1 . Time points are after repression by addition o f glucose. Cdc28p was used as a loading control.  kinetochore proteins tagged with G F P or V F P is well documented and is characterized b y a punctuate lobe adjacent to the nuclear side o f the spindle pole body. A s the spindle pole body is duplicated and separated, the sister kinetochores appear as two lobes that localize on the nuclear faces o f the spindle pole bodies (Measday et al., 2002; Pot et al., 2003). B y these criteria, R a d 6 1 - V F P did not display characteristic kinetochore localization, but instead showed a diffuse nuclear staining at all stages o f the cell cycle i n - 1 5 0 cells examined (Figure 2-8b and 2-8c). W e used N i c 9 6 - C F P to visualize the nuclear membrane and showed that R a d 6 1 - V F P was contained within the nucleus.  70  Untagged Control  Anti-MYC  Figure 2-8 - Rad61p localizes to the nucleus by indirect immunofluorescence and live cell  Merged  DAPI  b)  DIC  Merged  NIC96-CFP - red RAD61-VFP-green  c)  NIC96-CFP - red R A D 6 1 - V F P - green  DIC  imaging. A ) Indirect Immunofluorescence o f R A D 6 1 1 3 M Y C cells ( Y B C 1 9 8 ) . D A P I appears blue and M Y C appears red. - 1 5 0 cells were examined at different stages o f the cell cycle, the figure is a representative image. B ) Fluorescence microscopy on live cells ( Y B C 6 7 3 ) . These images are flattened Z-stacks (11 planes at 0.2 fim per plane). N i c 9 6 C F P appears red and R a d 6 1 - V F P appears green. - 1 5 0 cells were examined, the figure is a representative image. C) Increased magnification o f live cell imaging o f R a d 6 1 - V F P N i c 9 6 C F P cells.  A sub-fraction of Rad61p binds chromatin Rad61p localized to the nucleus but not in the distinct pattern characteristic o f kinetochore proteins. It was therefore o f interest to determine whether the nuclear localization o f Rad61p involved any binding o f chromatin. T o address this issue, two assays were employed. The first assay utilized the technique o f chromosome spreading whereby cells and nuclei are lysed with a detergent and spread onto a specially prepared microscope slide (that has been washed and boiled in 0.1% HC1). Chromatin is bound onto the slide and soluble proteins are washed away. The protein o f interest is visualized  71  using fluorescent secondary antibodies and D A P I is used to visualize chromatin. In chromosome spreads, R a d 6 1 - 1 3 M Y C p signal was found to co localize with D A P I i n > 100 nuclei examined and by this assay can bind to chromatin (Figure 2-9a, left panel). Ctf81 3 M Y C p is a chromatin bound protein and was used as a control (Figure 2-9a, right panel). The second assay used was chromatin purification, which involved centrifugation o f spheroplasted and lysed cells to isolate chromatin away from soluble proteins. In this assay, chromatin bound protein is purified with the chromatin i n the high speed centrifugation step. The amount o f Rad61p i n equal volumes o f the total mixture, the supernatant mixture (containing the soluble fraction o f the protein), and the pellet mixture (containing the chromatin bound fraction o f the protein) can then be assessed b y western blot analysis. W e used an antibody to acetylated H 4 as a control for a chromatin bound protein and an antibody to Carboxypeptidase Y ( C P Y ) as a control for a cytoplasmic protein. B y the chromatin binding assay a sub-fraction o f R a d 6 1 - V F P was isolated i n the chromatin fraction while a larger fraction o f the protein was contained i n the soluble fraction (Figure 2-9b). W e obtained similar results using R a d 6 1 - 1 3 M Y C p at different stages o f the cell cycle (data not shown). B y two different assays we found that a subfraction o f Rad61p i n the nucleus was binding to chromatin. It therefore appears that Rad61p is involved directly i n an aspect o f chromosome biology necessary for chromosome segregation. W e addressed the question o f the chromosomal locations o f Rad61p binding i n Chapter 3 using Chromatin Immunoprecipitation ( C M P experiments).  72  a)  Figure 2-9 - A sub-fraction of Rad61p binds chromatin.  RAD61-13MYCp - in Red DAPI - blue Rad61-VFP T  RAD61  S CP T  CTF8-13MYCp - in Red DAPI - blue  Untagged S CP  A ) Chromosome spreads on Rad611 3 M Y C strain ( Y B C 1 9 8 ) and Ctf81 3 M Y C strain ( Y P H 1 4 7 4 ) . A n t i - M Y C and secondary fluorescent antibodies were incubated at 1/1000 at room temperature. D A P I was contained i n the mounting media, D A P I appears blue, M Y C appears red. ~100 nuclei were examined and this is a representative figure. C t f 8 - 1 3 M Y C p was used as a control for binding to chromatin. B ) Chromatin Purification Experiment. Equal amounts o f total, supernatant and chromatin pellet fractions were loaded and anti-GFP (detecting the V F P tag) Westerns performed on strains expressing R a d 6 1 - V F P ( Y B C 6 7 1 ) o r a control untagged strain ( Y P H 4 9 9 ) . Acetylated Histone H 4 is used as a control for binding to chromatin and Carboxypeptidase Y ( C P Y ) is used as a control cytoplasmic protein.  is not involved in Homologous Recombination to repair DNA damage The major mechanism for repair o f D N A damage in budding yeast is b y  homologous recombination. In haploid cells, D N A repair by homologous recombination is conducted after S phase when there is an identical sister chromatid as a template for homologous recombination. W e were interested to see i f Rad61p was involved in  73  homologous recombination / repair because rad61A caused sensitivity to D N A damaging agents. The H O endonuclease in budding yeast is required for mating type switching, a mechanism whereby the mating type locus is switched to the silent mating type locus i n homothallic strains. The H O endonuclease induces a double strand break at a specific site i n the mating type locus and then the homologous recombination machinery repairs this double strand break using the silent mating type locus as the template. Mutants with defective homologous recombination cannot survive when the H O endonuclease is expressed as the double strand break cannot be repaired. When we overexpressed the H O endonuclease (using a galactose inducible promoter) i n w i l d type, rad61A, and rad52A (involved i n homologous recombination) strains, the rad61A and w i l d type strains were both able to grow whereas the rad52A strain was unable to grow (Figure 2-10a, compare the right hand panels, galactose media). M M S w i l l methylate D N A and cause D N A replication forks to stall and collapse and create double strand breaks. rad61A strains are sensitive to M M S (Figure 2-6a) and we were interested to see i f Rad61p's localization was affected b y M M S treatment and the generation o f double strand breaks. W e investigated the localization o f R a d 6 1 - V F P fusion protein i n M M S treated cells found that the localization remained similar to untreated cells (data not shown). There was still the possibility that RAD61 could be important, but not essential, for targeting the homologous recombination machinery to sites o f double strand break for repair. RAD52 is part o f a large epistasis group that functions i n homologous recombination. U p o n induction o f double strand breaks, it has been shown that Rad52p co-localizes with the double strand break sites (Lisby et al., 2003). Rad52p and the  74  homologous recombination machinery form "repair centres" that D N A double strand breaks w i l l co-localize with to be repaired.  W e tested whether deleting RAD61 could  affect the formation and localization o f R A D 5 2 - C F P foci i n response to the generation o f double strand breaks. W e used strains developed i n the Rothstein laboratory that contained RAD52-CFP,  an H O target site marked with L A C operator sequences, and a  constitutively expressed R F P - L A C repressor protein. W e transformed i n a vector that contained the H O endonuclease under the control o f a galactose inducible promoter (pJH 132).  W e induced double strand breaks b y inducing expression o f the H O endonuclease  by addition o f galactose and we monitored sites o f double strand breaks with R F P fluorescence (see Figure 2-10b, R F P and overlay panels, yellow and red arrows). W e also monitored Rad52-CFP foci formation (see Figure 2-10b, upper right panel, yellow arrow shows a Rad52-CFP foci, red arrow does not) and we counted the number o f cells that contained double strand break sites that had co-localizing Rad52-CFP foci. Figure 210b shows an example o f a co-localizing Rad52-CFP foci to the double strand break site (yellow arrows) and a double strand break site with no Rad52-CFP foci (red arrows). Table 2-4 shows the results; w i l d type and rad61A strains displayed similar numbers o f cells with double strand break sites visualized (28.3% to 31.1%) and had similar number o f R a d 5 2 - C F P foci co-localizing with those double strand break sites (52% to 53.8%).  75  a)  •?. * \  .  *  - «, >  • Overlay Green - RFP Red - CFP  •  •  RFP-LAC Repressor  Figure 2-10 - rad61A strain is not sensitive to overexpression of HO endonuclease and does not affect Rad52p foci formation. A ) W i l d type ( Y P H 4 9 9 ) , rad61A ( Y B C 2 0 4 ) , and rad52A ( Y B C 6 7 5 ) cells were transformed with a G A L - H O plasmid. C e l l cultures were grown overnight in selective media to maintain plasmid, and approximately equal amounts by absorbance were plated onto rich media either containing glucose as carbon source or galactose as the carbon source and allowed to grow at 25°C for 5 days. B ) Strains containing R A D 5 2 - C F P , as well as R F P - L A C repressor fusion protein and an H O cut site marked b y the L A C operator sequence ( Y B C 6 5 8 ) , were transformed with G A L - H O . Co-localization o f R A D 5 2 - C F P and the R F P marked H O cut site is induced after induction o f H O by addition o f galactose. Representative images from a strain that was also rad61A is shown ( Y B C 6 6 1 ) . R F P - L A C repressor appears green and Rad52-CFP appears red. The yellow  76  Figure 2-10 legend continued arrows mark an instance o f co-localization, while the red arrows mark an R F P - L A C repressor imaged double strand break that does not have a Rad52-CFP foci co-localized to it.  Thus, we concluded that Rad52-CFP foci formation and co-localization with double strand break sites is unaffected i n rad61A cells.  Table 2-4 Double Strand Break Induction - Rad52 foci formation is unaffected in rad61 A cells Strain  # of cells  # of DS break sites RFP foci  Rad52-CFP colocalizing with DS break sites  Wild type (YPH658), GALHO  346  98  51  28.3%  52%  65  35  31.1%  53.8%  Wild type (YPH658), GALHO as % rad61A (YBC661), GALHO rad61A (YBC661), GALHO as%  209  77  Discussion The rationale for finding proteins that genetically or physically interact with S g t l p at the budding yeast kinetochore was to identify novel proteins that played structural or regulatory roles important for kinetochore function. H i g h copy suppression screens have been used successfully to find biologically relevant protein partners (Connelly and Hieter, 1996; Kitagawa et al., 1999). It was reasonable to expect that by identifying high copy suppressors o f the temperature sensitivity phenotype o f sgtl-3 we could identify proteins important for S g t l p function, but we were unable to find proteins that were clearly linked to chromosome segregation to study. Neither o f the deletion mutants o f the uncharacterized O R F s that suppressed the sgtl-3 mutant missegregated a chromosome fragment and neither o f the deletion mutants in the two O R F s harboured phenotypes indicative o f a potential role i n chromosome segregation. The SOG2 and SOG3 O R F reports i n the Yeast Proteome Database indicate that the functions o f these proteins are unknown ( Y P D , www.incyte.com). W e also tested i f overexpressing Sog2p or Sog3p had an effect on sgtl-5 strains (with an S C F defect) to test i f Sog2p or Sog3p could be substrates o f the S C F but found no effect upon overexpression o f either protein (data not shown). U s i n g SGT1 as a bait in a genome wide two hybrid screen yielded results that were more biologically relevant to the study o f chromosome segregation. Finding many known interacting proteins o f S g t l p indicated that the screen was working but the fact that Rad61p could not be immunoprecipitated i n a complex with S g t l p indicates that the interaction may be transient in vivo or that the two hybrid result is an artifact that happened to isolate a protein involved i n chromosome segregation. The epitope tagged  78  constructs o f the two proteins may also interfere with their physical interaction. W e addressed the question o f potential Rad61p protein partners i n Chapter 3 using mass spectrometry approaches. Rad61p has all the characteristics o f a protein that is involved i n chromosome segregation. It is localized to the nucleus and a sub-fraction binds chromatin. The deletion mutant exhibits a chromosome missegregation phenotype, as do the truncation mutants found i n the original ctf mutant collection. Furthermore, the diploid null mutant displays a spindle checkpoint dependent cell cycle accumulation i n G 2 / M . W e did not see this phenotype i n the haploid null mutant. D i p l o i d null mutants displaying a defect (e.g. cell cycle progression delay) that is not seen i n a haploid null mutant has been observed before (Hyland et al., 1999). A diploid mutant may show more o f an effect compared to a haploid mutant because diploid cells have twice as many chromosomes i n the nucleus; phenotypes related to chromosome dynamics and segregation could be more accentuated as a result. The homozygous diploid null mutants also displayed resistance to intermediate levels o f the microtubule de-polymerizing drug, benomyl. B e n o m y l resistance has been demonstrated i n deletion mutants o f genes involved i n microtubule dynamics such as viklA (Manning et al., 1999) or kip3A (Cottingham and Hoyt, 1997). K i p 3 p is a kinesinrelated microtubule motor protein and V i k l p is a protein partner o f Kar3p, another kinesin-related microtubule motor protein. Microtubules i n kip3A mutant cells are longer and more stable compared to microtubules i n w i l d type cells (Cottingham and Hoyt, 1997). W e examined microtubules i n rad61A I rad61A diploid cells but found no difference compared to w i l d type diploid cells (data not shown). The resistance to  79  benomyl o f the rad61A / rad61A strain could be due to the delay i n progression through G 2 / M allowing cells time to stabilize microtubules and the mitotic spindle. W e tested for localization o f the protein and phenotypes that were indicative o f a role at the budding yeast kinetochore but consistently found that Rad61p did not appear to be a kinetochore component. The deletion mutant o f RAD61 is sensitive to D N A damaging agents (in particular to D N A double strand breaks) and we were able to confirm this phenotype i n our strain background. W e followed up on this data b y looking at aspects o f homologous recombination repair o f D N A but also found that Rad61p had no detectable defect i n Rad52p mediated homologous recombination repair. W e also did not detect differences i n Rad61p localization i n response to D N A damaging agents (such as M M S and ionizing radiation) and we do not believe that Rad61p is involved directly i n D N A repair. A n exact mechanism for Rad61p's role was not elucidated by direct tests for specific functions such as homologous recombination repair and kinetochore function. W e decided to explore different genomic and proteomic approaches i n order to understand the function o f Rad61p i n chromosome segregation. The reagents i n our laboratory and the techniques that have been pioneered in budding yeast were used to gain insight into the role o f Rad61p in chromosome segregation.  80  C H A P T E R 3:  Approaches to dissecting the role of Rad61p in chromosome segregation  81  Introduction To further characterize Rad61p and elucidate its biochemical functions, we decided to employ systematic genetic interaction technologies that have recently been developed. Specifically, we employed diploid-Synthetic Lethal Analysis on Microarrays ( S L A M ) to identify gene deletion mutants that were synthetically lethal in combination with rad61A.  Finding synthetic interactions on a genomic scale has helped to elucidate  the function o f genes by placing the query genes into clusters o f genes involved i n the same pathway (Tong et al., 2004; Pan et al., 2004). W e utilized epitope tagged constructs o f Rad61p which allowed us to immunoprecipitate the protein from budding yeast extracts and attempted to identify proteins or D N A that interacted with Rad61p. W e also cloned RAD61 into E. coli expression vectors that fused G S T to the N-terminus o f the protein. W e expressed this construct in E. coli and used yeast lysate to identify proteins that could bind to the purified GST-Rad61 protein. These experiments were used as approaches to dissect out the function o f Rad61p in chromosome segregation and represented initial screens that required further characterization and validation that allowed us to gain insight into Rad61p function.  82  Materials and Methods Yeast strains, growth conditions and media Yeast strains unless otherwise indicated were in the Y P H 499 strain background (S288C). In particular, the strains used i n the cohesion assays were i n the W 3 0 3 background. Yeast strains used i n Chapter 3 are listed i n Table 3-1. DED1 temperature sensitive alleles were obtained as gifts from Patrick Linder. The cloned mutant alleles were obtained on Y C p l a c l 11 L E U C E N plasmids. The plasmids were transformed into heterozygous DED1 / dedlA strains, which were sporulated to produce haploid strains with dedlA complemented by the DED1 allele containing plasmids.  Diploid-SLAM synthetic lethal screen The d i p l o i d - S L A M (Synthetic Lethality Analysis on Microarrays) was performed as previously described i n Pan et al., (2004). A RAD61 U R A 3 deletion cassette was transformed into the heterozygous deletion set pool. The d i p l o i d - S L A M was performed by X u e w e n Pan i n Jef Boeke's laboratory at Johns Hopkins. Random spore analysis was carried out using the query strain containing rad61 A::NATmated  to deletion mutant  strains o f interest. The diploids were sporulated and we selected for haploids using the Mat a specific HIS3 selectable marker (Tong et al., 2001). W e tested for growth o f the haploids on G418 plates (200 /ig/mL i n Y P D ) to select for query deletion strains, c l o n N A T plates (1 /xg/mL i n Y P D ) to select for rad61A, and G418 and c l o n N A T plates to select for the double mutants. The ratio o f colonies that grew on the different selection plates indicated synthetic interactions. If no colonies grew on the G418 c l o n N A T plates, a synthetic lethal interaction was scored.  83  Clustering analysis on diploid-SLAM results T w o dimensional hierarchical agglomerative clustering was performed on the data from the Tong et al., (2004) yeast genetic interaction database with the d i p l o i d - S L A M data added as a query. The query genes were clustered based on Average linkage and analysis and presentation o f the data was as described i n Tong et al., (2004). Clustering was performed b y Cluster 3.0 and visualized with Treeview 1.0.8.  Cohesion Assay Cohesion assays were performed as described i n Mayer et al., (2001). Cells were counted using a Zeiss A x i o p l a n 2 microscope using the lOOx objective lens.  ChlP-chip Assay ChlP-chip analysis was performed as previously described i n Horak et al., (2002). A strain containing R A D 6 1 - 1 3 M Y C tagged at the endogenous locus and Y P H 499 (wild type untagged control strain) were processed i n parallel, labeled and hybridized onto the same D N A chip. ChlP-chip analysis was performed by Anthony Borneman i n M i c h a e l Snyder's laboratory at Y a l e University. Confirmation P C R s were performed using primers to C E N 3 , C E N 1 6 and H M R (Measday et al., 2002).  84  Immunoprecipitation and GST purification Mass Spectrometry Co-immunoprecipitations were performed as described i n Measday et al., (2002) with the following modifications. IP samples were eluted from the 9E10 M Y C conjugated beads (Covance) by incubation at 65°C for 15 minutes. Eluted samples were trypsin digested and purified using a cation exchange column. Samples were then dried using Speed V a c Plus and tandem Mass Spectrometry was performed. Tandem mass spectrometry and analysis was performed as described i n Lee et al., (2004) by M a r k Flory in R u d i Aebersold's laboratory at the University o f Washington. F o r the GST-Rad61 purification we cloned R A D 6 1 into the p G E X vectors creating a GST-Rad61 fusion protein product. p G E X - R a d 6 1 was transformed into E.coli (BL21 strain) and expression o f the fusion protein was induced with 1 m M isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37°C for 3 hours. GST-Rad61 was contained i n the soluble fraction i n the bacterial cells and purified using glutathione agarose beads (Sigma). GST-Rad61 bound beads were then washed 4 times in 0.1% Triton in Phosphate Buffered Saline Solution ( T - P B S ) and subsequently incubated with yeast lysate overnight at 4°C. After washing 4 times with T - P B S , samples were eluted at 65°C for 15 minutes and subsequently treated the same as the EP samples used for tandem Mass Spectrometry analysis.  Co-Immunoprecipitations from Yeast Extract Cells Co-immunoprecipitations were performed as previously described i n Measday et al., (2002) using equal amounts o f protein.  85  Methods from Chapter 2 W e performed chromosome spreads, chromatin binding, fluorescence and indirect immunofluorescence microscopy, and F A C S analysis o f cells as described i n Chapter 2 Materials and Methods.  86  Table 3-1 List of yeast strains used in this chapter YPH499  M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al  trplA63  YPH500  M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al  trplA63  YBC285  M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 cep3-l M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 ctfl3-30 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 slkl9A::kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 chl4A::kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 sgtl-3: :LEU2 Mataura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 ctfl9A::TRPl M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 ctf3A::TRPl M a t a rad61A::HIS3 cin8A::kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 sgtl-5::LEU2 M a t a ura.3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 skpl-3::LEU2 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 skpl-4::LEU2 M a t a ade2-l trpl-1 canl-100 his3-ll,15 leu2::LEU2tetRGFP ura3::3xURA3tet0112 PDS1-13MYC-TRP1 M a t a ade2-l trpl-1 canl-100 his3-ll,15 leu2::LEU2tetRGFP ura3::3xURA3tetOU2 PDS1 -13MYC-TRP1 rad61A::HIS5 M a t a ade2-l trpl-1 canl-100 his3-ll,15 leu2::LEU2tetRGFP ura3::3xURA3tet0112 PDS1-13MYC-TRP1 rad61A::HIS5 M a t a trpl-1 :lacO-TRP-LEU2 his3-ll,15:lacR::GFP-HIS3 leu2-3,112 ura3-l ade2-l canl-100 M a t a trpl-1 :lacO-TRP-LEU2 his3-ll,15:lacR::GFP-HIS3 leu2-3,112 ura3-l ade2-l canl-100 rad61A::kanMX6 M a t a trpl-1 :lacO-TRP-LEU2 his3-ll,15:lacR::GFP-HIS3 leu2-3,112 ura3-l ade2-l canl-100 rad61A::kanMX6 Mata ade2 ura3 trpl leu2 SCC3-3HA-HIS3 Matctade2 ura3 trpl leu2 SMC1-6HA-HIS3 Mata ade2 ura3 trpl leu2 SMC3-6HA-HIS3 Mata ade2 ura3 trpl leu2 SCC2-6HA-HIS3 pep4A::LEU2  YBC289 YBC255 YBC329 YBC400 YBC423 YBC439 YBC676 YBC698 YBC699 YBC700 YPH1477 YBC534  YBC535  Y819 YBC564 YBC565 YBC583 YBC584 YBC585 YBC619  87  Sikorski and Hieter., 1989 Sikorski and Hieter., 1989 This study This study This study This study This study This study This study This study This study This study This study M a y e r et al., 2001 This study  This study M a y e r et al., 2001 This study This study K. K. K. K.  Nasmyth Nasmyth Nasmyth Nasmyth  Table 3-1 List of yeast strains used in this chapter, YBC688  YBC689  YBC690  YBC691 YBC692 YBC693 YBC694 YBC695 YH1444  YBC696  YBC697  YBC701  M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 CFIII (CEN3.L) URA3 SUP 11 containing YCp\acWl-dedl-56 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlAr.TRPl CFIII (CEN3.L) URA3 SUP 11 containing Y C p l a c l W-dedl-57 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 CFIII (CEN3.L) URA3 SUP11 containing Y C p l a c l W-dedl-58 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 rad61A::HIS3 containing Y C p l a c l W-dedl-51 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 rad61A::HIS3 containing Y C p l a c l 1 \-dedl-54 Mataura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 rad61A::HIS3 containing Y C p l a c l 1 \-dedl-55 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 rad61A::HIS3 containing Y C p l a c l 1 l-dedl-57 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 rad61A::HIS3 containing Y C p l a c l 1 l-dedl-58 M a t a ade2 trpl leu2 canl his3-l l,15::GFP(pAFS144,thermostable)-Lad-HIS3 leu2-3,112 ura3-l CEN 15(1.8 kb)-LacOURA3 M a t a ade2 trpl leu2 canl his3-l l,15::GFP(pAFS144,thermostable)-LacI-HIS3 Ieu2-3,U2 ura3-l CEN 15(1.8 kb)-LacOURA3 dedlA::kanMX6 containing Y C p l a c l W-dedl-51 M a t a ade2 trpl leu2 canl his3-ll,15::GFP(pAFS144,thermostable)-Lad-HIS3 leu2-3,112 ura3-l CEN15(1.8 kb)-LacOURA3 dedlA::kanMX6 containing Y C p l a c l W-dedl-51 M a t a ade2 trpl leu2 canl his3-ll,15::GFP(pAFS144,thermostable)-LacI-HIS3 leu2-3,112 ura3-l CEN15(1.8 kb)-LacOURA3 rad61A::HIS3  88  This study  This study  This study  This study This study This study This study This study Goshima and Yanagida., 2000 This study  This study  This study  Table 1 List of yeast strains used in this chapter, continued YBC603 YBC604 YBC607 YBC608 YBC611 YBC612 YBC649 YBC650 YBC589 YBC677 YBC678 YBC679 YBC680 YBC681 YBC682 YBC683 YBC684 YBC685  YBC686  YBC687  M a t a trpl ade2 ura3 leu2 RAD61 -13MYC-TRP1 SCC33HA-HIS3 M a t a trpl ade2 ura3 leu2 RAD61-13MYC-TRP1 SCC3-3HAHIS3 M a t a trpl ade2 ura3 leu2 RAD61-13MYC-TRP1 SMC16HA-HIS3 Mata trpl ade2 ura3 leu2 RAD61-13MYC-TRP1 SMC16HA-HIS3 M a t a trpl ade2 ura3 leu2 RAD61 -13MYC-TRP1 SMC36HA-HIS3 M a t a trpl ade2 ura3 leu2 RAD61-13MYC-TRP1 SMC36HA-HIS3 M a t a trpl ade2 ura3 RAD61 -13MYC-TRP1 SCC2-6HA-HIS3 pep4A::LEU2 M a t a trpl ade2 ura3 RAD61-13MYC-TRP1 SCC2-6HAHIS3 pep4A::LEU2 M a t a ura3 trpl ade2 leu2 rad61A::HIS3 SMC3-6HA-HIS3 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 AL Tl - VFP-kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 DED1-13MYC-kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trpl A 63 DED 1-13MYC-ka nMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 VPS3-13MYC-kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 VPS3-13MYC-kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 DEDl-13MYC-kanMX6 RAD61-VFP-kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 DEDl-13MYC-kanMX6 RAD61-VFP-kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 DEDl-VFP-kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trpl A 63 dedlA::kanMX6 CFIII (CEN3.L) URA3 SUP11 containing Y C p l a c l W-dedl-51 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 CFIII (CEN3.L) URA3 SUP11 containing Y C p l a c l W-dedl-54 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlAr.TRPl CFIII (CEN3.L) URA3 SUP11 containing Y C p l a c l W-dedl-55  89  This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study  This study  This study  Results  rad61A  has genetic interactions with kinetochore a n d cohesion gene mutations  To potentially gain insight into Rad61p function, we tested if rad61A displayed genetic interactions with mutants known to be defective in different aspects of chromosome biology. We mated a rad61A strain with strains carrying mutations in genes involved in kinetochore or cohesion function (Table 3-2), sporulated the double heterozygous diploids, and assessed the phenotypes of double mutant haploids following tetrad dissection. rad61A displayed synthetic interactions with central kinetochore gene mutations (ctf3A, chUA, ctf 19A), a microtubule associated protein gene (kar3A) and also with cohesin and cohesin loading factor mutations (scc2-4, smc3-42). Table 3-2 rad61A genetic interactions Strain  Genetic Interaction  rad61A ceo3-1 (YBC285)  Viable  rad61 A ctf13-30 (YBC289)  Viable  rad61 A skp1-3.4 (YBC699.700) Viable rad61 A sot1-3,5 (YBC400.698) Viable rad61 Acin8A (YBC676)  Viable  rad61 Aslk19A (YBC255)  Viable  rad61 A ctf3 A (YBC439) rad61 A ndd 0-42  CSL 2 SL  rad61 A kar3 A  SL  rad61 Achl4A (YBC329)  CSL  rad61 A ctf19 A (YBC423)  CSL  rad61 A scc2-4  SL  rad61 A smc3-42  SL  90  1 C S L - conditional synthetic lethality - the double mutant can survive at 2 5 ° C but the restrictive temperature of a temperature sensitive allele is lowered or for two non-essential gene deletion mutants, there is lethality at higher temperatures for the double mutant. 2 Synthetic Lethality - Two nonessential gene deletion mutants are inviable in combination or a nonessential gene deletion and a temperature sensitive allele in an essential gene are inviable in combination.  CEP3, CTF13,  NDClO-umei  kinetochore proteins SKPI, SGT1 - inner kinetochore and SCF proteins  SLK19, CTF3, CHL4, CTF 19central kinetochore CIN8, KAR3 - microtubule motors SCC2 - Cohesin loading protein SMC3 - subunit of core cohesion complex  Testing specific mutants for genetic interactions with rad61A does not provide a comprehensive data set, and is therefore limited. To address this issue we performed a d i p l o i d - S L A M screen to attempt to identify all non-essential genes i n the genome wide gene deletion set that had synthetic interactions with rad61A. The d i p l o i d - S L A M screen requires the generation o f a double heterozygote diploid " p o o l " that is heterozygous for both the deletion mutant o f interest (in this case rad61A) and each o f the deletion mutants in the budding yeast deletion set (refer to Figure 3-1 for a schematic o f the d i p l o i d - S L A M technique). Systematic diploid generation is accomplished by transformation o f a pool o f the entire heterozygous diploid deletion collection with a rad61A cassette with an auxotrophic marker ( U R A 3 ) . The heterozygous deletion mutant diploid product is selected for and then induced to sporulate. After sporulation, an auxotrophic marker, HIS3, that is only expressed i n M a t a specific cells, is used to select for haploid cells. Further selection using the markers for the deleted genes is also used to isolate double mutant haploids. Haploid double mutant combinations that exhibit synthetic lethality or slow growth i n the pool o f all possible double mutant combinations with the query strain w i l l be selectively lost from the population upon outgrowth. Each o f the deletion mutants in the deletion collection contain unique molecular tags, "barcodes", which can be used to assess i f the population contains that deletion mutant. Genomic D N A was prepared from populations that were rad61A or RAD61  and the "barcodes" amplified b y P C R ,  labeled with C y 3 (rad61A) or C y 5 (RAD61 )  and both hybridized to a D N A microarray  +  +  that contains the anti-sense sequences to the "barcodes". The relative abundance o f the "barcodes" o f each o f the deletion mutants i n the two pools indicates the synthetic interaction.  91  •~ I HETEROZYGOUS D I P L O I D S MAI  Hi:  XXX/m.\  KtnMX  TRANSFORM^ OAML • LEW? • HfAtm • HIS} • CANIR SELECT Oft CONVERTIBLE HETEROZYGOUS SC LEU  otnoios  .=?  S  MATalu  X X X / mti  SS  MrrMX  CANUcants LEU2-MFA1pr-HI$3 CONVERTIBLE HETEROZYGOUS  OIPLOiD POOL  g  I HETEROZYGOUS OIPLOrOS  POOL  NWW« —. YtGUylgn  S B xxx SS  / m i  URA3 kaoMX  CA/Vf/canU LEU2-MFAlpr-MS3  SPOftULATE ,  V«'»HAPLOIDS  SELECT ON MAGIC MEDIUM IMM  •—-MAT* =>/fj|lA  UfiAJor — Y F G f *  • =  ktnUX  UHA  = a « w f A -  LEU2-UfA1pr-HIS3  CONTRCH  EXPEKIMEN1AI  MM » F O A  MM -URA  Single mutants  DouMe mutants  ANALYZE WITH MICROARRAY  rift R e d T A G » p o t * indiane Y K O *  synthetic lethal with query Y K O Orange T A G spots suggest synthetic fitness defect  From Pan etal., (2004)  F i g u r e 3-1 - Schematic o f the d i p l o i d - S L A M technique. See the text for details and also Pan et al., (2004). A ) Transformation o f the heterozygous diploid pool with HIS3 under the M F A 1 promoter to allow selection o f haploids. B ) Heterozygous diploid pools are transformed with yfgA::URA3 and sporulated. Haploids are selected and the relative abundance o f strains is analyzed with a microarray.  92  See Table 3-3 for the results o f the screen. W e performed random spore analysis on a subset o f the results and found i n all instances that the synthetic interactions were reconfirmed (6/6 interactions tested by random spore analysis were re-confirmed, see Table 3 for the deletion mutants tested). W e were also able to detect a synthetic interaction between rad61A and all four deletion mutants that had previously been directly tested (ctf3A, kar3A, chl4A, and ctfl 9A), which gave more confidence in the results o f the screen. A s expected from the directed double mutant analysis, rad61A displayed synthetic interactions with the central kinetochore gene mutations and also with cohesin and cohesin loading factor mutations, and with other factors that are involved i n the establishment o f sister chromatid cohesion. The results o f the d i p l o i d - S L A M were clustered with the data from Tong et al. (2004) using a clustering program first developed for use with microarray transcription data. The program was used to cluster binary synthetic interaction data, with 0 representing no synthetic lethality and 1 representing synthetic lethality. rad61A clustered with sccl-73  fa temperature sensitive mutant o f the  core cohesin complex member S c c l p ) as well as CHL1, a D N A helicase that is involved i n sister chromatid cohesion (Figure 3-2).  93  Table 3-3 Diploid SLAM Results RANK  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  GENE NAME (or ORF)  SGOl CHL4 RSC1 CSM3  • * Tested and confirmed with Random Spore Analysis CTF4  Genes involved in chromosome segregation  BRE2 MCM16 CTF18  Cohesion  MGA2 YPL017C YLR111W CTF8 DUN1 YDR018C RAD27  LST4 LSM1 DCC1  94  Query Strains  Deletion Mutant Array  ICTF8 CTF4 HRC1 P0L32 RAD 2 7  ELG1 DDC1  Figure 3-2 - Clustering of d-SLAM genetic interactions with genome wide SGA screens from Tong et al., 2004. RAD61 clusters with sec J-73 and CHL1 (boxed genes). Query strains form columns with the deletion mutant array as rows. Red indicates a synthetic lethal interaction. Clustering was performed by Cluster 3.0 and visualized with Treeview 1.0.8.  95  Rad61p is involved in sister chromatid cohesion and potentially in establishment RAD6Vs  genetic interactions clustered with genes involved i n cohesion so we  were interested i n testing the possibility that Rad61p was important for sister chromatid cohesion. To test this possibility we performed a sister chromatid cohesion assay. Cells that have chromosome locations marked with T E T or L A C operator sequence arrays are arrested i n either G l phase (before D N A replication has occurred) using the mating pheromone alpha-factor, or i n G 2 / M (after D N A replication) using a microtubule depolymerizing drug, nocodazole. Sister chromatids are visualized using G F P tagged L A C repressor or T E T repressor fusion proteins that w i l l bind to the operator arrays. Precociously separated sister chromatids w i l l appear as two distinct G F P signals whereas sister chromatids that remain together w i l l appear as one signal (two G F P "dots" versus one "dot" i n nocodazole arrested cells). The alpha-factor arrested cells serve as a control for aneuploidy; cells should have only one G F P signal before D N A replication. When we performed this assay, we found that rad61A strains displayed a sister chromatid cohesion defect. In w i l d type strains, the percentage o f cells with two distinguishable G F P signals i n G 2 / M was consistently 9%. In rad61A strains, the percentage was approximately 18%. Presumably the 9% o f cells that display two G F P signals in w i l d type cells arrested with nocodazole represent a basal rate o f breathing o f the paired chromatids. This assay was performed for two sites on chromosome arms as well as a site close to the centromere; the magnitude o f the defect is comparable at all three sites (Figure 3-3a). To determine i f a chromatin bound fraction o f Rad61p could be potentially binding at a chromosomal sequence known to be enriched for cohesin binding, we  96  performed a ChIP assay using P C R primers flanking that site. In the ChIP assay, proteinD N A interactions are fixed using formaldehyde treatment (to cross-link p r o t e i n - D N A interactions). The cells are then lysed and the cell lysate is sonicated i n order to shear D N A to 300 to 500 base pairs i n length. The protein o f interest that has been epitope tagged is immunoprecipitated using antibodies to the epitope tag and the immunoprecipitated mixture is incubated at 65 °C i n order to reverse the formaldehyde cross-linking; D N A is subsequently isolated b y phenol extraction. Analysis o f the D N A fragments that immunoprecipitated with the protein o f interest can be assessed by P C R using primers flanking the region o f interest. Although we could clearly see cohesion D N A i n the S m c l p control ChIP, no enrichment o f binding o f Rad61p to the cohesin binding site was observed (Figure 3-3e). It was possible that Rad61p may be interacting with the cohesin complex that is not on chromatin and we tested this possibility b y coimmunoprecipitation experiments performed on yeast extracts. W e performed A n t i - H A IPs using yeast extracts from strains that carried either endogenously tagged or SMC1-6HA, RAD61-13MYC.  or SMC3-6HA  SCC3-3HA,  individually or i n combination with endogenously tagged  W e could not detect any association o f Rad61p w i t h Scc3p, S m c l p or  Smc3p (Figure 3-3b; see lanes 2, 6, and 10). Thus, Rad61p does not appear to interact directly with the core cohesin complex at cohesin binding sites. To test whether or not Rad61p was involved i n loading o f cohesin onto chromatin, we performed chromosome spreads i n both w i l d type and rad61A strains that expressed an epitope tagged cohesin component (Smc3-6HAp). Cohesin is loaded onto chromatin i n G l (after release from alpha-factor arrest) and dissociates i n anaphase. rad61A  strain  background had no effect on the kinetics o f loading or the dissociation o f cohesin (Figure  97  3-3d). W e examined - 5 0 nuclei at each o f the time points i n Figure 3-3d and representative images o f S m c 3 - 6 H A p on or off chromatin for w i l d type and rad61A strains are shown. To test i f there could be an interaction between Rad61p and the complex that loads the cohesin complex onto chromatin, we performed a coimmunoprecipitation assay on yeast extracts prepared from two strains that had endogenously tagged R a d 6 1 - 1 3 M Y C p and Scc2-6HAp. W e found that there was no detectable association between the two proteins i n extracts prepared from both strains tested (Figure 3-3c, see lanes 6 and 8). A new class o f non-essential genes that seem to be involved in the establishment o f sister chromatid cohesion has recently been described. M a n y o f the genes have a defect in sister chromatid cohesion that is comparable to that observed i n rad61A mutant strains (Mayer et al., 2001; Mayer et al., 2004; Warren et al., 2004). It is believed that the timing o f cohesion establishment is during or right after D N A replication. Rad61p by virtue o f having a sister chromatid defect and not being involved either as a core cohesin complex member or the loading o f cohesin, may similarly be involved i n the establishment o f cohesion.  98  Sister chromatid cohesion assay  a)  i^HHIBIiBHHflilHBIi^H  o.a Ik  15  ~  10  *  5  t  i  iI. Nxodasle  Nocodaale  il,,  i | alpha-factor  alpha-factor arrest  alpha-factor arrest  r e l e a s e - 3 0 min  release - 75 min  c) Anti-HA IP  Anti-HA IP 6 7 8  alpha-factor arrest  factor Chromosome XV, 1.8 kB from CEN  Chromosome V, 35 Kb from CEN  4 5  rad6m  alpha-  j alpha-factor Chromosome IV, right arm  1 23  SMC3-6HAp  Rad61-13MYC  9101112 Rad61-  Scc2  13MYC  6HA  RAD61-13MYC Untagged  Scc2-6HA #1  TSIPT  SIP  1 2 3 4  5  TS  #2 IPT  S  TOT  IP  TOT  IP  SMC3-6HA TOT  IP  IP Scc2-6HAp  1/ RAD61-13MYC SCC3-3HA TOT 21RAD61-13MYC SCC3-3HA IP 3/ SCC3-3HA TOT 4/ SCC3-3HA IP 5/ RAD61-13MYC SMC1-6HATOT 6/ RAD61-13MYC SMC1-6HA IP 7/SMC1-6HATOT 8/SMC1-6HAIP 9/ RAD61-13MYC SMC3-6HA TOT 10/ RAD61-13MYC SMC3-6HA IP 11/SMC3-6HATOT 12/SMC3-6HAIP  6  7 8 9 1011  12 Cohesin binding site - C h r o m o s o m e V - 5 4 9 7 Kb  1 2  3  4  5  6  7  8  Control site - P O L 1 - Y N L 1 0 2 W Rad61-13MYCp  1/RAD61-13MYCTOT 2/RAD61-13MYCIP 3/ SCC2-6HA TOT 4/ SCC2-6HA IP 5/ RAD61-13MYC SCC2-6HA TOT 61RAD61-13MYC SCC2-6HA IP 7/ Same as 5, different isolate 81 Same as 6, different isolate  Figure 3-3 - rad61A strains display a cohesion defect that is likely a result of defective establishment. A ) Sister Chromatid Cohesion Assay. We counted the number o f cells with 1 or 2 G F P signals. The error bars represent 200 cells counted in 2 different experiments. N o error bars - represents 200 cells counted. We tested three different chromosomal loci, one on Chromosome V (35 kb from C E N ) ( Y P H 1 4 7 7 , Y B C 5 3 4 , 5 3 5 ) , Chromosome I V arm (Y819, YBC564,565), and Chromosome X V (1.8 kb from C E N ) ( Y P H 1 4 4 4 , Y B C 7 0 1 ) . B ) A n t i - H A Co-IP on yeast extract expressing Rad611 3 M Y C p and S c c 3 - 3 H A p (lanes 1 and 2) ( Y B C 6 0 3 ) , S c c 3 - 3 H A p (lanes 3 and 4) ( Y B C 5 8 3 ) , R a d 6 1 - 1 3 M Y C p and S m c l - 6 H A p (lanes 5 and 6) ( Y B C 6 0 7 ) , S m c l - 6 H A p (lanes 7 and 8) ( Y B C 5 8 4 ) , R a d 6 1 - 1 3 M Y C p and S m c 3 - 6 H A p (lanes 9 and 10) ( Y B C 6 1 1 ) , or S m c 3 - 6 H A p (lanes 11 and 12) ( Y B C 5 8 5 ) . Total and IP samples were run on gels for Western blot using A n t i - H A or A n t i - M Y C with the strains only expressing a single tag as negative controls.  99  Figure 3-3 Figure legend continued C) A n t i - H A Co-IP performed on yeast extracts from strains expressing R a d 6 1 - 1 3 M Y C p (upper panel lanes 1 to 3, bottom panel lanes 1 and 2) ( Y B C 1 9 8 ) , S c c 2 - 6 H A p (upper panel lanes 4 to 6, bottom panel lanes 3 and 4) ( Y B C 6 1 9 ) , and two strains expressing R a d 6 1 - 1 3 M Y C p and Scc2-6HAp (upper panel lanes 7 to 12, bottom panel lanes 5 to 8) ( Y B C 6 4 9 , 650). The two single tagged strains were used as negative controls. Western blots were performed using A n t i - M Y C and A n t i - H A . D ) Chromosome Spreads ( Y B C 5 8 5 , S m c 3 - 6 H A ) and ( Y B C 5 8 9 , S m c 3 - 6 H A rad61A). W e performed chromosome spreads using A n t i - H A primary and a fluorescent secondary ( G A N - F ) at 1/1000 dilutions. S m c 3 - 6 H A appears red and D A P I appears green. A t each o f the time points we examined ~50 nuclei, and representative images are shown. E ) ChIP P C R assays on strains expressing R a d 6 1 - 1 3 M Y C p ( Y B C 1 9 8 ) , S m c 3 - 6 H A p ( Y B C 5 8 5 ) , or untagged control strain (YPH499) using primers for a cohesin binding site and POL1 as a noncohesin binding site control.  By ChlP-chip analysis Rad61p associates with regions around centromere DNA In an effort to understand Rad61p's role i n binding chromatin we sought to determine i f the chromatin binding occurred at specific loci on chromosomes. W e performed a Chromatin Immunoprecipitation followed by microchip analysis (ChlP-chip experiment) i n collaboration with Michael Snyder's laboratory at Y a l e University. The ChIP technique (formaldehyde fixation, lysis, sonication, immunoprecipitation, reversal o f cross-link and D N A extraction) is similar (see Materials and Methods) to that used to assess the binding o f Rad61p to the putative cohesin binding site. In ChlP-chip, the detection o f binding sites involves hybridization o f immunoprecipitated D N A to sequences on a D N A chip. Genomic D N A that has been sheared and isolated is labeled and used as a probe on a D N A microchip (containing intergenic sequences o f the yeast genome). A parallel ChIP is performed on an untagged control strain to produce genomic D N A that is labeled and used as a control for non-specific D N A binding i n the EP. The D N A samples from both ChIP experiments are labeled with different fluorescent markers and hybridized to the same D N A chip. Binding sites are determined based on the  100  hybridization ratio o f the R a d 6 1 - 1 3 M Y C p immunoprecipitated D N A sample over the untagged control immunoprecipitated D N A sample with a threshold that must be met i n order to be scored as a binding site (Horak et al., 2002; Anthony Borneman, personal communication). The data is then presented on a map o f the yeast chromosomes with diamonds depicting the binding sites with the diamonds stacked i f the binding sites cluster. Four separate immunoprecipitation experiments were performed. W e found that there was specific binding to a majority o f the centromere regions (12 o f the 16 chromosomes) in budding yeast with little other specific binding sites i n the rest o f the genome (Figure 3-4a). Confirmation P C R s were performed using template D N A from R a d 6 1 - 1 3 M Y C p tagged strains or from untagged strains and there did appear to be enrichment at C E N D N A sequences compared to n o n - C E N sequences such as HMR, TEC1 and MGA1 (Figure 3-4b, upper panels are C E N D N A sequences, compare left and right lanes. Lower panels are the control negative sequences). The ChlP-chip analysis and confirmation P C R s were performed i n M i c h a e l Snyder's laboratory at Y a l e University; when we performed confirmation P C R s using C E N 3 and HMR sequences we found that using our protocols, a known kinetochore protein, N d c l 0 - 1 3 M Y C p , could immunoprecipitate C E N D N A while R a d 6 1 - 1 3 M Y C p could not (Figure 3-4c, compare EP lanes o f R A D 6 1 - 1 3 M Y C and N D C 1 0 - 1 3 M Y C ) . The contradictory results are likely due to differences i n the protocol employed. The fixation time o f the ChEP-chip analysis was longer and may have cross-linked proteins that are further from the core kinetochore. Differences i n sonication treatment o f the yeast lysate could shear D N A to different average sizes and this would affect the sequences that could be detected i n the analysis. The nuclear localization o f Rad61p does not correspond to  101  known kinetochore proteins (Figures 2-8b and 2-8c) and we conclude that Rad61p's preferential localization to centromeres is transient, sub-stochiometric, or indirect through other proteins.  Chrom I,  i.  Chrom IV  B, t  Chrom V, VI, VII  Chrom VIII, IX, X  Chrom XI, XII  " *•  at  1  ki  i t  it  iii  1  t  *'  • CEN  > >  t  ttt  l  l  , i  t 1  t  t l.  1  YBC198YPH499 YBC198YPH499 YBC198YPH499  CEN16  t i t  tt  Chrom XV, XVI  Uhtagged  t i t i t  1  t  RAD6113MYC  J  i .  Chrom XIII, XIV  b)  i  RAD61- Un13MYC tagged  M  c)  #tt  YBC198  Y P H 4 9 9 YIP412  RAD61-13MYC  RAD61- Un13MYC tagged  Total  CEN2  IP  Untagged NDC10-13MYC  Total  IP  Total  IP  CEN3  T IP T IP T IP HMR  Figure 3-4 - Rad61p by ChlP-chip analysis is specifically enriched at Centromere DNA. A ) Chromosomes I to X V I are visualized with diamonds representing spots o f Rad61p specific binding over the w i l d type (over the threshold S D o f 4) Centromeres are indicated by the black dots. B ) ChEP P C R assays using strains expressing Rad611 3 M Y C p or an untagged control strain were performed using Primers to C E N III, X V I , and control regions HMR, TEC1 and MGA1. C) Chromatin Immunoprecipitation Assay using primers to C E N 3 and H M R . Strains expressing N d c l O - 1 3 M Y C p or Rad611 3 M Y C p , and an untagged control strain were used for the ChIP assay. Increasing concentrations o f the template D N A sequence were used for the total and IP samples i n the 3 strains for the C E N 3 P C R . H M R was used as a control for n o n - C E N background binding.  102  Interaction between Rad61p and Dedlp A s an alternative approach to dissecting the function o f Rad61p, we explored different methods o f finding proteins that interacted with Rad61p. W e performed immunoprecipitation experiments on budding yeast extracts from a R A D 6 1 - 1 3 M Y C strain. Eluted IP samples were digested with trypsin, purified and tandem mass spectrometry was performed. Dr. M a r k Flory i n Rudi Aebersold's laboratory at the University o f Washington performed the tandem mass spectrometry analysis. W e were consistently unable to identify more than 2 peptides o f Rad61p i n the elution mixture. W e decided to modify our approach and expressed a GST-Rad61 construct i n E. coli, purified the construct with glutathione agarose beads and then incubated the beads with budding yeast lysate i n order to find potential interactions. W e were able to recover approximately 200 peptides o f Rad61p as w e l l as other peptides specifically i n the G S T Rad61 experiments versus G S T alone ( A l t l p , D e d l p , Vps3p, Kar2p, S n u l 3 p , T f p l p , and T u b l p ) (see Table 3-4). W e tested the top three proteins ( A l t l p , D e d l p and Vps3p) for interactions with Rad61p by co-immunoprecipitation experiments using epitope tagged constructs o f Rad61p and each o f the potential interacting proteins and we found a reproducible interaction between Rad61p and D e d l p (Figure 3-5a, see lane 3 and lane 15 o f the panels). W e did not detect interactions between Rad61p and Vps3p (Figure 3-5a, lane 9 i n the upper panels, and lane 18 i n the bottom panel) or with Rad61p and A l t l p (Figure 3-5b, see lane 6). A peptide o f D e d l p had also been identified previously i n the initial R A D 6 1 - 1 3 M Y C IP experiments.  103  Table 3-4 GST-Rad61 purified protein incubated with Protein  # of peptides  ALT1  9  VPS3  5  DED1  5  KAR2  2  SNU13  2  TFP1 *  2  TUB1  2  * Common Contaminant  D e d l p is an essential protein and is an R N A helicase o f the D E A D - b o x family with a documented role i n the initiation o f translation (lost et al., 1999; Chuang et al., 1997). D e d l p also has a nuclear role (discussed below) but its documented localization is cytoplasmic (as would be expected from a gene involved i n protein synthesis). D e d l p has also been implicated as part o f the spliceosome (Burckin et al., 2005) and as a potential interactor with Cdc28p (the C D K i n budding yeast) and therefore it may have additional functions inside the nucleus (Honey et al., 2001). W e were able to show that D e d l - 1 3 M Y C p and Rad61-VFPp epitope tagged constructs could interact when expressed at endogenous levels from their endogenous loci i n budding yeast cells (Figure 3-5c, see lane 6). In fission yeast there is intriguing evidence that D e d l p could be posttranslationally modified and that translation o f other proteins could be affected in response to different forms o f cellular stress ( L i u et al., 2002). W e decided to check the  104  interaction between Rad61p and D e d l p when cells have undergone D N A damage induced b y M M S . W e found that the interaction between Rad61p and D e d l p did not detectably change i n cells that had been treated with M M S (data not shown).  a)  YBC678  YBC680  DED1-13MYC GAL-HAHA-RAD61  T  GAL control  VPS3-13MYC GALGAL HA-RAD61 control  S IP T S IP T S IP T S IP VPS3-13MYC DED1-13MYC  HA-RAD61 1 2  3 4 5 6 7  8  DED1-13MYC Gal-HA-RAD61 Tot Sup IP  9 1011 12  VPS3-13MYC Gal-HA-RAD61 Tot Sup IP HA-RAD61  13  14 15  YPH499  b)  16  17 18  YBC677 ALT1-VFP  GALGAL HA-RAD61 control  T  IP  T IP  GALGAL HA-RAD61 control  T  IP  T  IP HA-RAD61 ALT1-VFP  7  8  YBC682  c)  Tot Sup IP  Ded1-13MYC Rad61-VFP Tot Sup IP Ded1-13MYC Rad61-VFP  Figure 3-5 - Protein-protein interactions of Rad61p and Dedlp and unconfirmed interactions of Rad61p with Altlp and Vps3p. Figure legend on the next page. 105  Figure 3-5 - Protein-protein interactions of Rad61p and Dedlp and unconfirmed interactions of Rad61p with Altlp and Vps3p. A ) Strains expressing D E D 1 - 1 3 M Y C with a G A L - H A - R A D 6 1 plasmid (lanes 1 to 3 and 13 to 15), D E D 1 - 1 3 M Y C with a G A L control plasmid (lanes 4 to 6 i n the upper panels), V P S 3 - 1 3 M Y C with a G A L - H A - R A D 6 1 plasmid (lanes 7 to 9 in upper panels, and lanes 16 to 18 i n lower panel), or V P S 3 - 1 3 M Y C with a G A L control plasmid (lanes 10 to 12 i n upper panels), were grown i n galactose (2%) to induce H A - R a d 6 1 p expression. Yeast extracts were then prepared and IPs carried out with A n t i - M Y C beads. Western blots were performed and A n t i - M Y C or A n t i - H A used to visualize proteins. L o w e r panel is re-running o f the samples from the upper panels. B ) Untagged strains with G A L - H A R A D 6 1 plasmid (lanes 1 and 2) or with a G A L control plasmid (lanes 3 and 4), and strains expressing A L T 1 - V F P with a G A L - H A - R A D 6 1 plasmid (lanes 5 and 6), or with a G A L control plasmid (lanes 7 and 8), were grown i n 2 % galactose to induce H A - R a d 6 1 p expression. Yeast extracts were then prepared and A n t i - H A IPs were carried out. A n t i G F P and A n t i - H A Westerns were performed on the samples. C ) Yeast extracts from strains expressing R a d 6 1 - V F P (lanes 1 to 3), or R a d 6 1 - V F P and D e d l - 1 3 M Y C p (lanes 4 to 6) at endogenous levels were prepared and A n t i - M Y C IPs performed. A n t i - G F P and A n t i - M Y C Western blots were performed to detect proteins.  Dedlp binds chromatin and has a role in genome integrity D e d l p has well documented roles i n initiation o f translation and at the spliceosome but because o f its interaction with Rad61p we were interested to see i f D e d l p could potentially have other roles in the nucleus. Because Rad61p binds chromatin, it was o f interest to determine i f there could be a fraction o f D e d l p that bound chromatin. B y chromatin purification experiments (performed i n duplicate on two independent clones expressing D e d l - 1 3 M Y C p ) , D e d l - 1 3 M Y C p binds to chromatin (Figure 3-6a). Acetylated H 4 was used as a control for chromatin binding and C P Y was used as a control cytoplasmic protein; both o f the controls behaved as expected and D e d l - 1 3 M Y C p contained a chromatin bound fraction. Chromosome spreads also showed that D e d l - 1 3 M Y C p could bind chromatin and that the staining looked to be spread across chromatin with no discemable foci (data not shown). When we looked at  106  the localization o f all the D e d l p b y live cell microscopy using D e d l - V F P , we found that the majority o f the protein localized to the cytoplasm (Figure 3-6b).  YBC678 679  YPH499  DED1-13MYC Untagged #3 #6 Tot Sup C P Tot Sup C P Tot Sup C P DED1-13MYC  H4 CPY  b)  YBC684  DIC  Figure 3-6 - Dedlp binds chromatin but the majority of the protein is in the cytoplasm. A ) Chromatin purification experiments were done as described. Equal amounts o f total, supernatant and chromatin pellet fractions o f two D e d l - 1 3 M Y C p expressing strains, and an untagged control strain, were run and a Western blot using A n t i - M Y C was used to visualize the amounts o f D e d l p in each fraction. Acetylated H 4 and C P Y were used as controls for chromatin and cytoplasmic proteins. B ) L i v e cell fluorescence microscopy on D e d l - V F P cells. Cells were immobilized by low melting point agarose (see Materials and Methods). W e examined - 1 0 0 cells and this a representative image using one focal plane.  107  DED1 is an essential gene i n budding yeast and certain temperature sensitive alleles have been shown to cause both translation and splicing defects at the nonpermissive temperature. W e were interested to see i f temperature sensitive alleles o f DED1 may have defects i n chromosome segregation at permissive temperatures and also whether or not they had cohesion defects when shifted to non-permissive temperatures. Six temperature sensitive alleles o f DED1 on yeast centromere plasmids were obtained from Patrick Linder's laboratory (dedl-51, dedl-58).  dedl-54, dedl-55, dedl-56, dedl-57, and  W e transformed the plasmids into diploid strains that had one copy o f the  DED1 gene deleted and also contained a non-essential chromosome fragment that contained the SUP11 marker (in order to assess the amount o f chromosome missegregation o f the strain containing the dedl allele). W e also transformed the plasmids into another diploid strain that had one copy o f DED1 gene deleted and that was homozygous null for RAD6L  After sporulation o f the diploids, we isolated haploids that  contained the dedl A complemented by the dedl temperature sensitive alleles on the plasmids. The restrictive temperatures o f the strains containing dedl A complemented b y the different dedl mutant alleles are shown i n Table 3-5. A l l strains containing a combination o f mutant alleles o f dedl and rad61A were able to grow. However, the restrictive temperatures o f strains containing two temperature sensitive alleles, dedl-51 and dedl-57, i n combination with rad61A, were lowered, indicating a genetic interaction between the mutations o f the genes (Table 3-5). To assess i f the dedl mutant alleles caused strains to lose chromosomes, we compared the red/white sectoring (indicating the loss o f the non-essential chromosome fragment) between the strains containing the dedl mutant alleles and a w i l d type strain. The strains  108  containing the dedl alleles did not have strong sectoring phenotypes at 25 °C although dedl-55 containing strains at higher temperatures showed an increased amount o f red sectors compared to the w i l d type strain (Table 3-5 and Figure 3-7). dedl-55 mutant strains contained 238 half-red sectors i n a population o f 6018 colonies counted (3.95%) versus 18 half-red sectors i n a population o f 4113 w i l d type colonies counted (0.44%) representing an increase o f approximately 9x that o f w i l d type strains.  Table 3-5 DED1 mutants and their sectoring phenotypes DED1 Mutant  Mutation  Restrictive Temperature  Sectoring at 35°C (or 33°C if lethal at 35°C)  Genetic Interaction with  RAD61  ded1-51  YBC685  L403 S M to V in Cterminal  35°C  Wild type  CSL  ded1-54 (686)  L403 S  37°C  Wild type  Nl  ded1-55 (687)  M 183 R  37°C  ~9x wild type  Ni  ded1-56 (688)  P 526 L  37°C  Wild type  —  ded1-57 (689)  H 430 R  35°C  Wild type  CSL  ded1-58 (690)  I 404 T  37°C  Wild type  Nl  Nl No Interaction CSL Conditional Synthetic Lethality  109  Wild Type YPH1020  ded1-55 YBC687  F i g u r e 3-7 - dedl-55 mutants sector at 35°C. There is an increase number o f red sectors in the dedl-55 strain compared to w i l d type. B y half-sector analysis, dedl-55 strains lose the chromosome fragment at a rate ~ 3 to 4x that o f w i l d type.  In order to assess i f the chromosome missegregation phenotype o f the dedl mutant alleles could be caused by sister chromatid cohesion defects, strains were constructed that had the dedl -51 allele covering the dedl A with a chromosomal locus marked i n order to assess precocious sister chromatid separation. Cells were arrested with a-factor and released into media containing nocodazole either at 3 0 ° C (permissive temperature) or 37°C (restrictive temperature). A t 15 minute time points cell cultures were shifted from 3 0 ° C to 37°C, i n order to inactivate D e d l p function at different points. W i l d type cells and the dedl-51 strain arrested as expected i n a-factor and nocodazole at 30°C (Figure 3-8a) but the dedl-51 strain at 37°C had a delay in progression through S phase. W e found that increasing the time at 30°C allowed a larger fraction o f cells to duplicate their D N A and be subsequently arrested in G 2 / M by nocodazole (Figure 3-8a, compare rows 3 to 8). The binding o f D e d l p to chromatin (Figure 3-6a) suggests that  110  this S phase progression delay could be a direct consequence o f inactivating D e d l p . Alternatively, the delay could be caused b y aberrant transcription or translation o f G l or S cyclins similar to a temperature sensitive allele found i n hamster B H K 2 1 cells, ts E T 2 4 cells, w i t h a mutation i n D D X 3 X (a D E A D - b o x gene), which caused a G l / S arrest with a decrease i n the m R N A o f cyclin A (Fukumura et al., 2003). A mutant defective i n the translation initiator eIF4E i n budding yeast, w h i c h caused a G l to S phase progression defect, was also rescued by expression o f Cln3p (Danaie et al., 1999). There were very few large budded cells with duplicated D N A i n the dedl-51 strains arrested with nocodazole at 37°C. Assessing precocious sister chromatid separation was therefore difficult. Preliminary results indicate that there may be a defect i n sister chromatid cohesion i n dedl-51 strains at 37°C as approximately half o f the large budded cells contained 2 G F P signals (46 / 90; 51%) with the 2 G F P signals in the same "mother" compartment (Figure 3-8b).  Ill  a)  b)  LOG  Alpha-factor  1  1  DIC  Nocodazole 30°C  Nocodazole 37°C  jJi  — GFP  Nocodazole 15 min 30°C Then to 37°C  Nocodazole 30 min 30°C then to 37°C  Nocodazole 45 min 30°C Then to 37°C  L U LLJ  Nocodazole 60 min 30°C Then to 37°C  Figure 3-8 - dedl-51 strains have a cell cycle defect and potential sister chromatid cohesion defect. A ) F A C S profiles o f w i l d type (YPH1444) and a dedl-51 ( Y B C 6 9 6 ) ts mutant arrested i n alpha-factor and released either into YPD-nocodazole at 30°C or 37°C. A t 15 minute time points cell cultures were also shifted from 30°C to 37°C. G l and G 2 peaks are labeled. B ) A n example cell from the sister chromatid cohesion assay showing a large budded cell containing 2 G F P signals both in the same "mother" compartment o f the cell.  112  Discussion In an effort to understand the functions o f Rad61p in vivo we used both genetic and biochemical (proteomic) approaches. The d i p l o i d - S L A M technique allowed the identification o f all the deletion mutants in the genome wide deletion set that were synthetically lethal with rad61A.  Direct tests for synthetic interactions had been  performed previously with rad61A and deletion mutants o f genes important for kinetochore and sister chromatid cohesion function and all four interactions that had been identified previously (ctfA, chl4A, kar3A, ctf 19A) were also identified i n our diploidS L A M screen. There were no false negatives from the d i p l o i d - S L A M results compared to the direct tests. Clustering the results o f the synthetic lethality profile o f rad61A led us to test for cohesion defects in rad61A strains because rad61A clustered next to mutants i n cohesion genes. W e indeed observed a cohesion defect but were unable to show any physical interaction o f Rad61p with either the core cohesin complex or with the Scc2/4 cohesin loading complex. B y chromosome spreads, cohesin loading, association and dissociation from chromatin appear unaffected i n rad61A strains. Rad61p may be involved i n establishing sister chromatid cohesion. To investigate the possibility that Rad61p was binding to specific chromosomal loci, we collaborated with Michael Snyder's laboratory and performed ChlP-chip analysis on strains containing R a d 6 1 - 1 3 M Y C p expressed at endogenous levels. U s i n g the ChlP-chip assay, Rad61p bound specifically at regions clustered around centromeres. Extensive work purifying the components o f the yeast kinetochore i n recent years has not identified Rad61p as a component o f the budding yeast kinetochore (De W u l f et al., 2003; Measday and Hieter, 2004). W e also did not see the typical localization pattern  113  seen for kinetochore associated proteins when we examined localization o f Rad61p through the cell cycle (Figure 2-8b, 2-8c). It is possible that Rad61p is binding C E N D N A transiently and/or this transient binding is difficult to detect because o f Rad61p binding to general chromatin (Figure 2-9a). The fixation time used i n the ChlP-chip assay is longer than that used i n our follow-up experiments (Figures 3-4b and 3-4c) and this may have an effect i n detecting interactions that are less direct and occurring through other proteins at the kinetochore. Rad61p may be associated at the periphery o f the budding yeast kinetochore. The binding o f Rad61p to regions clustered around C E N D N A is also reminiscent o f the cohesin complex association with centromere regions (Lengronne et al., 2004; G l y n n et al., 2004). The biochemical approach that we initially undertook to find proteins that physically interact with Rad61p was to perform immunoprecipitations on extracts from yeast expressing endogenous levels o f R a d 6 1 - 1 3 M Y C p . The eluted samples were purified and the proteins digested with trypsin, with the tryptic peptides subjected to tandem mass spectrometry analysis. W e were consistently unable to purify more than 2 peptide fragments o f Rad61p even when we scaled the culture to five litres. Rad61p may be labile and the immunoprecipitation and washing combined with the trypsin digests and purification may have caused much o f the protein to be degraded. W e did however isolate one peptide fragment o f D e d l p in one o f our experiments, but because o f the low number o f Rad61p peptides it was not immediately followed up on. W h i l e we were able to see intact R a d 6 1 - 1 3 M Y C p by western blotting o f our IP samples (data not shown), there also appeared to be degradation bands suggesting loss o f sample due to degradation.  114  The alternative approach that we decided to pursue was that o f producing a large amount o f GST-Rad61 protein i n E. coli, purifying the protein using glutathione agarose beads and then incubating the beads with yeast lysate. W e performed this experiment with a G S T vector construct alone as a negative control for yeast proteins that would bind G S T and the glutathione agarose beads. This approach helped us to isolate hundreds o f Rad61p peptide fragments and also specific peptides that were present i n the GST-Rad61 and not i n the G S T alone control experiment. Testing the top three results confirmed that Rad61p did indeed bind to one o f these proteins, D e d l p , and that this interaction occurred during a normal cell cycle (the IP was performed using log phase cells) with both proteins expressed at their endogenous levels. W e had performed the initial characterization o f the binding o f D e d l p and Rad61p using an over-expressed H A - R a d 6 1 fusion protein and it was important to show that the proteins could interact when they were both expressed at their endogenous levels in yeast extracts. D e d l p belongs to a large family o f putative R N A helicases that have nine conserved amino acid motifs including the characteristic A s p - G l u - A l a - A s p ( D E A D ) box (Rocak and Linder, 2004). A l o n g with the D E A H and D E x H - b o x families they form a large super-family o f helicases that are highly conserved and are present i n bacteria, yeast, and humans (Abdelhaleem et al., 2003). The D E A D - b o x helicases use the energy released from A T P hydrolysis to cause re-arrangements o f R N A including unwinding R N A duplexes and displacing proteins from R N A molecules (Linder, 2003; Rocak and Linder, 2004). There are 27 D E A D - b o x proteins i n budding yeast and they have been implicated i n many aspects o f R N A metabolism including R N A synthesis, translation, r R N A processing, ribosomal bio-synthesis and R N A degradation (Rocak and Linder,  115  2004). Purified D e d l p has been shown to have R N A helicase activity in vitro and also to be able to displace proteins from R N A molecules without unwinding the R N A duplex (that is without acting as a helicase); both activities occur i n an A T P dependent manner (Fairman et al., 2004). D e d l p ' s role in initiating translation is well established and a dedl cold sensitive allele shows defects i n global protein synthesis when shifted to nonpermissive temperature. A decline i n polyribosome levels occurs almost immediately upon shift to lower temperatures consistent with a direct role o f D e d l p i n translation initiation (Chuang et al., 1997). The putative role for R N A helicases at translation initiation is unwinding R N A duplex structures at the 5' Un-Translated Region ( 5 ' - U T R ) allowing the ribosome to scan the m R N A to the A U G (lost et al., 1999). Localization o f D e d l p has also been shown to be cytoplasmic, which was re-confirmed i n our strain background, and consistent with its role i n m R N A translation. D e d l p also has a nuclear role as a member o f the spliceosome and also as a Cdc28 interacting protein isolated by mass spectrometry analysis o f purified Cdc28p (Honey et al., 2001). DED1 was originally isolated as a suppressor o f the prp8-l mutation, a gene involved in splicing (Jamieson et al., 1991). U s i n g splicing-sensitive microarrays ( D N A chips that contain sequences that correspond to intronic sequences and to sequences at 5' and 3' U T R s ) , dedl temperature sensitive alleles were found that clustered with genes involved in m R N A translation and also with genes involved in splicing (Burckin et al., 2005). D e d l p could also bind to small nuclear R N A s (snRNAs) indicative o f a role in splicing (Burckin et al., 2005). It was concluded that D e d l p had functions both in translation and i n splicing and that any link between the two functions would need further characterization.  116  What could be the potential role o f Rad61p and D e d l p together i n chromosome segregation? It seems likely that any role w o u l d be direct, as both proteins bind chromatin and can physically interact. The genetic interactions between mutant alleles o f dedl and rad61A and also o f rad61A and mutants i n kinetochore and sister chromatid cohesion function, are consistent with a direct role i n chromosome segregation. The biochemical function o f the two proteins has not been elucidated and there remains the possibility that the role o f D e d l p i n chromosome segregation is i n affecting the expression level o f a protein important for chromosome segregation. D e d l p has been implicated in both translation and splicing and it is possible that its role i n those two processes could contribute to chromosome loss through indirect action on genes involved i n chromosome segregation. For example, Prpl 7, a gene coding for a protein o f the spliceosome was initially characterized as Cdc40 and mutant strains displayed cell cycle defects. The cell cycle defects o f a prpl 7 mutant could be suppressed b y deleting the intron of ANC1, a gene that is implicated i n cell cycle control (although the exact function o f A n c l p is unclear) (Dahan and Kupiec, 2004). Approximately 3.8% o f yeast genes contain introns (the majority having only one intron) but these yeast genes are estimated to account for 27% o f the total m R N A transcripts i n typical cells (many ribosomal proteins contain an intron as w e l l as genes coding for actin and tubulin) (Ares et al., 1999). U s i n g microarray chips that contain intronic sequences (the splicing-sensitive chips mentioned above), researchers have recently been able to determine effects o f deleting specific genes involved i n splicing and w h i c h genes containing introns are affected (Burckin et al., 2005). Specific effects on different intronic genes by deletion o f different members o f the splicing machinery (and also o f  117  genes involved i n m R N A metabolism) can be assessed. Some o f the genes that have introns that are interesting in terms o f potential chromosome segregation defects and/or cell cycle control defects include GLC7, a phosphatase that de-phosphorylates I p l l p substrates, MOB1, part o f the mitotic exit network, TUB1 and TUB3, coding for alphatubulin, ACT1 coding for actin, and PH085  involved i n cell cycle control  (Saccharomyces Genome Database, S G D ) . D e d l p ' s role i n translation initiation could be another potential way that D e d l p has an indirect role on chromosome segregation. Proteins that are involved i n cell cycle regulation, kinetochore function and cohesion could be potentially reduced i n a dedl hypomorph mutant. In the fission yeast S. pombe, the homologue o f D e d l p has been found to associate with Cdc2 (the C D K i n fission yeast) and with C h k l (a protein kinase that is involved i n response to D N A damage) ( L i u et al., 2002). It was shown that D e d l p could be post-translationally modified i n response to heat shock and to depletion o f carbon (but not to D N A damage) and it was concluded that D e d l p may be playing a role in affecting protein translation and synthesis i n response to cellular stress ( L i u et al., 2002). A n attractive idea would be that D e d l p is affecting translation o f proteins that are needed to respond to the cellular stress o f damage. DED1,  In budding yeast, a mutant allele o f  dedl-18, has been found to impair brome mosaic virus ( B M V ) R N A 2 translation  while still maintaining general translation o f other m R N A and allowing growth (Noueiry et al., 2000). D e d l p could either have a direct effect i n selecting m R N A for translation or the helicase activity o f the mutant allele could be decreased below a threshold required to unwind R N A secondary structure i n the B M V R N A 2 m R N A to allow translation initiation (Noueiry et al., 2000).  118  C o u l d Rad61p have a role with D e d l p in translation initiation or m R N A splicing? The rad61A deletion mutant does not have growth defects at any o f the temperatures tested and it seems unlikely that Rad61p would have a function i n initiating translation, although it may have a role i n translating a subset o f proteins that are important for chromosome segregation. Testing the expression levels o f a subset o f proteins that are known to be involved in kinetochore or cohesion function could be attempted. In this regard we have checked protein levels o f cohesin complex members and some members o f the kinetochore complexes and have found no detectable difference i n expression levels between w i l d type and rad61A cells (data not shown). A role for Rad61p in splicing could be addressed by using splicing-sensitive chips to check i f there were splicing defects i n rad61A cells versus w i l d type. Different components o f the splicing machinery appear to have potentially different effects on specific subsets o f yeast genes that have introns. A global approach to assessing all the yeast genes with introns would be the best way to assess any splicing defect that may exist in RAD61 deletion mutants. The other possibility is to check protein expression o f genes that have documented roles in cell cycle regulation and chromosome dynamics. From the experiments that were performed on D e d l p it seems likely that D e d l p has roles other than splicing in the nucleus. A fraction o f D e d l p binds chromatin, but it is unclear what function this chromatin binding D e d l p fraction is performing. It would be very informative to perform ChlP-chip experiments on D e d l p and characterize any particular chromosomal locations to which D e d l p is binding. It would be interesting to see i f D e d l p is binding the same locations as Rad61p, which would suggest that the chromatin binding fractions o f the proteins are interacting. D e d l p ' s chromatin binding  119  can also be assessed in rad61A strains to investigate i f D e d l p chromatin binding is dependent on Rad61p. A t this point it is unclear whether Rad61p is interacting with D e d l p on chromatin or whether there is some small fraction o f Rad61p i n the cytoplasm that cannot be detected by microscopy. The conditional synthetic lethality between rad61A and temperature sensitive alleles o f dedl points to the importance o f the interaction between the two proteins. Preliminary data showing sister chromatid cohesion defect i n dedl-51 may point to a mechanism for the chromosome missegregation phenotype.  120  CHAPTER 4: Conclusions and Discussion  121  Approaches to Identifying novel components involved in chromosome segregation In an effort to identify novel components that are involved i n chromosome segregation, and i n particular, kinetochore function, we used SGT1 as a genetic entry point. S g t l p is a protein partner o f S k p l p and plays a role with S k p l p at the budding yeast kinetochore and the S C F complex. W e were interested i n finding proteins that could play a role i n regulation o f the kinetochore and since S g t l p plays a role i n activating the C B F 3 complex, we reasoned that it would be a suitable entry point. W e attempted two large scale screens, a high copy suppression screen and a genome wide two-hybrid screen, and we identified a novel factor i n chromosome segregation, Rad61p, that is apparently not directly involved in S g t l p function, but is important i n sister chromatid cohesion.  Genome Wide Two-hybrid screen oiSGTl  identifies RAD61  The approach that led to the identification o f Rad61p as a novel factor i n chromosome segregation was a genome wide two-hybrid screen using SGT1 as the "bait". W e were able to identify genes encoding known protein partners o f S g t l p , including S k p l p and F-box proteins such as Cdc4p, G r r l p and Met30p. These results gave us confidence that we were identifying proteins that were biologically relevant to S g t l p function. W e were especially interested in Rad61p, because the deletion mutant o f the gene displayed a chromosome missegregation phenotype. W e were unable to detect an interaction between S g t l p and Rad61p b y co-immunoprecipitation experiments using logarithmically growing yeast extracts and we concluded that the interaction was transient, sub-stochiometric, or a potential artifact o f the two-hybrid screen. W e found  122  when we performed a ChlP-chip analysis with long fixation times using formaldehyde to cross-link D N A - p r o t e i n interactions that Rad61p could bind preferentially to regions close to centromeres. The two-hybrid interaction may represent an indirect interaction between Rad61p and S g t l p through the kinetochore and proteins indirectly associated with the kinetochore. In addition to a chromosome missegregation phenotype, rad61A diploid strains also displayed a G 2 / M progression delay that was dependent on Mad2p. W h e n we investigated the morphology o f the cells, we found that rad61A strains contained more cells with short spindles and the nucleus at the neck, compared to w i l d type cells. This was indicative o f a delay i n progressing through the metaphase to anaphase transition consistent with the delay depending on Mad2p. Mutants displaying a similar phenotype have been found b y mutation o f genes encoding proteins involved i n processes such as kinetochore, microtubule, and sister chromatid cohesion function (Hyland et al., 1999; M a y e r et al., 2001). A s we continued to characterize Rad61p, we found that it was localized to the nucleus and also bound chromatin. These characteristics were consistent with a direct role i n chromosome segregation. rad61A mutants are also sensitive to D N A damaging agents, especially to D N A damaging agents that cause a double strand break (Game et al., 2003; this study). W e explored the possibility that Rad61p could be involved i n homologous recombination mediated D N A repair but found that it was not necessary for repair o f a specific double strand break and that it was not necessary for D N A "repair centres" as determined b y Rad52p foci formation.  123  Rad61p and its role in sister chromatid cohesion U s i n g the d i p l o i d - S L A M technique to find synthetic interactions o f rad61A, and combining that data with the large database o f synthetic interactions uncovered b y Tong et al., (2004), we were able to cluster rad61A with sec 1-73 and chllA,  two known players  in sister chromatid cohesion. When we examined rad61A strains, we indeed found that they had a sister chromatid cohesion defect. Rad61p belongs to a group o f non-essential proteins that when deleted have a moderate sister chromatid cohesion defect. M a n y o f these proteins have been provisionally categorized as proteins involved i n establishing sister chromatid cohesion during or immediately after D N A replication because they are not part o f the core cohesin complex, are not needed to maintain cohesion once established, and are not involved i n loading cohesin onto chromatin. The D N A damage sensitivity o f radSIA mutant strains may be the result o f the moderate defect i n sister chromatid cohesion. Several other non-essential gene deletion mutants exhibiting moderate sister chromatid cohesion defects are also sensitive to D N A damaging agents, including chllA,  ctjSA, dcclA,  and ctf4A (Bennett et al., 2001). W e  also found that sec 1-73 and smc3-42 containing strains, with defects in the core cohesin components, were sensitive to bleomycin and M M S (data not shown). Recent studies have shown that sister chromatid cohesion is required for post-replicative D N A double strand break repair (Sjogren and Nasmyth, 2001; Strom et al., 2004; U n a l et a l , 2004). Cohesin is recruited to the sites o f double strand break and this recruited cohesin is functional for sister chromatid cohesion (Strom et al., 2004). H o w sister chromatid cohesion is generated outside o f S phase remains unclear. Establishment o f cohesion may simply depend on proximity o f the sister chromatids provided i n S phase  124  immediately behind the replication fork and i n G 2 and M (at double strand break sites that have recruited cohesin) by sister chromatid cohesion on the arms established during S phase (Unal et al., 2004). There may be other factors that are important and it would be interesting to see i f deleting RAD61 or any o f the non-essential genes involved i n sister chromatid cohesion could affect cohesin recruitment or the establishment o f sister chromatid cohesion at sites o f double strand breaks. Once established, sister chromatid cohesion likely facilitates homologous recombination repair between sister chromatids by tethering the broken ends together and ensuring the right template sequence is available. Rad61p does not interact with the cohesin complex (or with chromosomal loci known to be cohesin binding sites) and does not interact with Scc2p, part o f the cohesin loading complex. It has also not been identified as a member o f any o f the non-essential complexes that are important for sister chromatid cohesion (Hanna et al., 2001; M a y e r et al., 2001; Mayer et al., 2004; Warren et al., 2004). Identifying proteins that interact with Rad61p could shed light onto its role i n sister chromatid cohesion.  Rad61p and Dedlp interaction W e incubated a large amount o f purified GST-Rad61 recombinant protein with yeast lysate and found specific proteins that bound to GST-Rad61 and not to the G S T control. W e tested the top three "hits" as determined by the number o f peptides that were identified by mass spectrometry, and found that we could reproduce the interaction between Rad61p and D e d l p in cells expressing endogenous levels o f the two proteins. D e d l p is an R N A helicase o f the D E A D - b o x family, a large family o f A T P dependent R N A helicases that function i n R N A metabolic processes such as splicing, ribosome bio-  125  synthesis, and translation initiation. W e showed that a portion o f D e d l p binds to chromatin in addition to the documented localization i n the cytoplasm. The functional significance o f the interaction between D e d l p and Rad61p is supported by the observed genetic interactions between rad61A and two temperature sensitive alleles o f  DEDL  Dedlp and its role in chromosome segregation One o f the temperature sensitive alleles o f DED1 (dedl-55) exhibits an increase in chromosome missegregation at a semi-permissive temperature, as evidenced b y a nine fold increase i n the rate o f loss o f a chromosome fragment at 35°C. The dedl-55 mutation (M183R) is not in a conserved motif but is in a predicted /3-sheet (Tanner et al., 2003) and the mutation may disrupt the secondary structure o f the protein. The data demonstrating that dedl-51 has a sister chromatid cohesion defect may account for the chromosome missegregation phenotype. It is possible that D e d l p could be having an indirect effect on sister chromatid cohesion through m R N A translation or splicing affecting the expression level o f one or more proteins that are necessary for sister chromatid cohesion, but we believe this is unlikely because o f its localization onto chromatin and the genetic interactions o f dedl temperature sensitive alleles with rad61A. The clustering o f rad61A with sccl-73 and chllA ( S c c l p is a core component o f cohesin and C h l l p is a D N A helicase important for sister chromatid cohesion) based on synthetic lethal interactions with deletion mutants o f the yeast genome wide deletion set suggests a direct function for Rad61p in sister chromatid cohesion. The genetic interaction between dedl ts alleles and rad61A suggests that Rad61p and D e d l p binding in yeast cells is functionally significant. Clustering analysis placing uncharacterized genes into w e l l  126  known pathways has identified novel components o f those pathways (Tong et al., 2004). D e d l p could be directly involved with Rad61p in sister chromatid cohesion i n addition to its roles i n translation and splicing. There are examples o f other proteins that play multifunctional roles in cells; for example, Noc3p was initially found to be required for r R N A processing and pre-ribosome maturation (in the nucleolus) but also subsequently demonstrated to be a chromatin binding protein important for the initiation o f D N A replication (Zhang et al., 2002). D e d l p could be playing multiple roles i n the budding yeast cell i n translation, splicing, and sister chromatid cohesion.  RNA Helicases in Human Cancers A recent survey using the PSI-protein B L A S T program identified 36 members o f the D E A D - b o x family and 14 members o f the closely related D E A H - b o x family i n the Homo Sapiens non-redundant peptide sequence database (Abdelhaleem et al., 2003). The functions o f the putative D E A D - b o x R N A helicases that have been investigated are the same as the D E A D - b o x R N A helicases i n budding yeast; namely R N A metabolic processes such as m R N A splicing, ribosome biogenesis, translation, m R N A export and m R N A stability (Abdelhaleem et al., 2003). M a n y o f the D E A D - b o x family members remain un-characterized but two important characteristics o f the D E A D - b o x family members that have been investigated have been described; there is a dysregulation o f expression i n cancer and an involvement i n differentiation (Abdelhaleem, 2005). Human R N A helicases have been found to be overexpressed in colorectal cancer (DDX5 (p68) (Causevic et al., 2001), DDX6 (Nakagawa et al., 1999)), as w e l l as other cancers and down-regulated i n acute lymphoblastic leukemia (DHX32 ( D E A H - b o x  127  family member), (Abdelhaleem, 2002)). There is also evidence that human R N A helicases can interact with proteins implicated i n cancer. For example, D D X 2 interacts with Pdcd4, a protein with transformation-suppressing activity (and Pdcd4's overexpression can inhibit transformation), and this activity is linked to its binding to D D X 2 (Yang et al., 2003a; Y a n g et al., 2003b). DDX10 was found to be involved i n a leukemia-associated chromosomal translocation I n v l I(pl5q22) generating an in-frame fusion o f the DDX10 to NUP98.  There are many more examples o f R N A helicase links  with cancer (see the recent review from Abdelhaleem, (2004) for a list o f the human R N A helicases and the cancer-related evidence). While the closest human homologues to D e d l p i n humans ( D D X 3 X , D D X 3 Y , and D D X 4 1 ) do not have documented roles i n cancer and tumourigenesis, D e d l p is also highly homologous to the other D E A D - b o x family members (especially i n the nine conserved domains). The accepted role o f human R N A helicases i n R N A processing can contribute to tumourigenesis in at least three ways, through altering splicing, transcription and translation o f m R N A (Abdelhaleem, 2004). Altering expression o f oncogenes and/or tumour suppressor genes could lead to tumourigenesis and altering R N A helicase activity (either b y mutations, deletions o f the gene, or by overexpression) could be one o f the "hits" needed for tumour progression. W e have shown evidence that a temperature sensitive mutation i n budding yeast DED1 can cause an increase i n the rate o f loss o f a non-essential chromosome fragment. There is no clear evidence to show that this phenotype is a direct consequence o f D e d l p function on chromatin. The chromosome missegregation phenotype o f the dedl ts allele may be the result o f altering the expression o f a gene that is involved i n chromosome segregation. Finding the gene that  128  -I  is responsible for the loss o f the chromosome fragment based on its altered expression i n the DED1 mutant would provide a rationale for how mutating R N A helicases could affect processes such as chromosome segregation. A connection between R N A helicases and genes important for chromosome segregation fidelity would be an interesting avenue o f research to explore i n colorectal cancer cell lines that are both aneuploid and overexpressing R N A helicases. C o u l d R N A helicases be playing a more direct role i n chromosome segregation and tumourigenesis? Could there be effects other than on R N A metabolism? A recent report showed that some o f the R N A "helicases" (including D e d l p ) could displace proteins from R N A duplexes without "unwinding" the R N A duplex; that is without its R N A helicase activity (Fairman et al., 2004) indicating that novel activities o f these proteins can still be uncovered. Recombinant Dpb9p (a budding yeast D E A D - b o x protein involved in ribosome biogenesis) was also found to have D N A helicase activity in vitro although the physiological significance o f this is unclear ( K i k u m a et al., 2004). However, D e d l p does not show D N A helicase activity (Patrick Linder, personal communication). There is evidence that D e d l p can physically interact with a checkpoint kinase, C h k l p , i n fission yeast extracts ( L i u et al., 2002) and that DED1 has genetic interactions with CDC2 ( C D K i n fission yeast). In budding yeast D e d l p has been identified as a protein interactor with Cdc28p. W e also identified D e d l p as a protein interacting with a nuclear localized protein, Rad61p. A fraction o f both Rad61p and D e d l p binds chromatin. These results suggest a potential role for D e d l p on chromatin but definitive proof o f a biochemical function has not been obtained.  129  RAD61 and potential human homologues A B L A S T search using the Rad61p protein sequence reveals two human sequences with weak homology to Rad61p. L O C 5 7 8 2 1 is a putative human protein and has had little characterization performed and is the most significant human protein found from a Rad61p protein query (over a region o f 230 amino acids, there is 18.4% identity and 29.4% similarity). L O C 5 7 8 2 1 has been identified as one o f the putative homologues o f ctf genes (specifically o f RAD61) that are candidates for sequencing i n colorectal cancer cell lines with the CEN phenotype (Karen Yuen, personal communication). A P E (AKT-Phosphorylation Enhance), K I A A 1 2 1 2 , is 19.3% identical and 29.4% similar to Rad61p over a region o f 190 amino acids. A P E has been recently shown to be a novel interactor o f Protein Kinase B ( P K B ) , a kinase involved as a key mediator o f signal transduction with a wide variety o f substrates, all o f which have the consensus motif R X R X X ( S / T ) (Anai et al., 2005). A P E may function in D N A synthesis and apoptosis i n cooperation with P K B .  Conclusion Rad61p was initially identified as a potential protein partner o f S g t l p based on a genome wide two-hybrid screen using SGT1 as the "bait". Rad61p and S g t l p did not detectably interact i n co-immunoprecipitation experiments performed on yeast extracts and Rad61p did not appear to be an S C F substrate. RAD61 is not essential, but deleting the gene has consequences for the fidelity o f chromosome segregation, increasing the rate o f loss o f endogenous chromosomes to ~100x that o f w i l d type strains. The chromosome  130  Figure 1 - Putative Human homologues of Rad61p in Human Cells a)  RAD61 LOC57821 RAD61  31 IfDlSsViPJgVSlTlssllAD^ 105 fflES^ElQKfflLEEEEDEEWRSM|Kl|C^KLfflQNQEPVND^EBKLKF^QL|DL 89 @NV@|l|SSSg[rS MA^KlJEKJgSgFNFgDGSKASKR^RgTYQK^ANgTS  LOC57821 RAD61  162 E I P B --LgjTfflsaY^NEgNgGKlSQLC@SNDF|Q|i^fflLS|TNGSCEENK139 S i E @ D V Q D ^ ^ l 3 M ^ E ^ S l S K S N D | N E F I § K L P R | D ^ ^ N K f L E N E M K M D D S  LOC57821 RAD61  214 --DRpHLVE^GEELLNLQD^SlGILPPlfcANSlSNfflpQQLLPRSSfflSSVfaGT 195 IENNggRTSggKggGKFRTILgNK^ KEfJEIMGE§\/gQKANTLSLN§ADNgNA  LOC57821 RAD61  268 K ^ D s B A K l f f l A V T H S S T ^ P f A H 290 247 E^GLgSTNBJYNELKNM^TgKg 269  b) RAD61 KIAA1212  60 H p f ^ A Q D l s S ^ D G A N E K P s B Q - - L D | K R N | Q B K i l T S S g S - M 0 F f W D E I O j S A F f S F L D 671 BN^BLKKflaLD^BKMLTFOLF^LEKENfioLDwE^tfElSRRNV^^LKcSRr^MAoBnLEBKETj  RAD61 KIAA1212  117 731  RAD61 KIAA1212  172 KgPRADDD|LNK|l^NE|KMDDSIENNSIRTSl^]KKYGKFRTILl|KNf^gElSGE 78 8 DgEMENQTgQKMjE@LKflSSKRLEQLEKENK§I^QETSQLEKDKK§LE^^KRgRQ  RAD61 KIAA1212  228 844  GHKASKR^RRTYQ^DANinss|raPDVffl--raElsIT|HNEFrasiRraiYSD|NEF|L EgEKEQJ^GLELL^SFKKgERg^SY^LSlaNQRB^  E VjEofflANfflSLfflADNsSAf^^GLfl 252 Q A I J I j g D T ^ E E H v K I G p j ^ ^ N K g 868  F i g u r e 1 - P u t a t i v e Human homologues o f Rad61p i n Human C e l l s . A) Rad61p and LOC57821 are 18.4% i d e n t i c a l and 29.4% s i m i l a r near t h e N - t e r m i n a l o f each p r o t e i n . B ) Rad61p and KIAA1212 (APE) a r e 19.3% i d e n t i c a l and 29.4% s i m i l a r i n a l i m i t e d r e g i o n , n e a r t h e N - t e r m i n a l o f Rad61p and i n t h e m i d d l e o f APE.  131  missegregation phenotype o f rad61A strains is the result o f a defect i n sister chromatid cohesion and Rad61p may play a role i n sister chromatid cohesion establishment based on its chromatin localization. Rad61p physically interacts with a D E A D - b o x R N A helicase, D e d l p , and this interaction is functionally significant as evidenced b y genetic interactions between temperature sensitive alleles o f DED1  and rad61A. D e d l p is a  multifunctional protein with characterized roles i n initiating translation and also as a member o f the spliceosome. W e found that a sub-fraction o f D e d l p binds to chromatin and a mutant allele o f DED1  loses a non-essential chromosome fragment at a rate ~9x  that o f w i l d type strains at a semi-permissive temperature. Preliminary data also points to a sister chromatid cohesion defect for dedl-51 strains pointing to a possible mechanism for the chromosome missegregation phenotype. Taken together, these results suggest that Rad61p and D e d l p cooperate together on chromatin i n a process that is necessary for sister chromatid cohesion. Human homologues o f Rad61p and D e d l p would therefore be candidates to screen for mutations that may be causing the C I N phenotype i n human cancer cell lines.  132  REFERENCES Abdelhaleem, M . (2002) The novel helicase homologue D D X 3 2 is down-regulated i n acute lymphoblastic leukemia. LeukRes 26: 945-954. Abdelhaleem, M . (2004) D o human R N A helicases have a role i n cancer? Biochim Biophys Acta 1704: 37-46. Abdelhaleem, M . 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