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

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I D E N T I F I C A T I O N O F N O V E L F A C T O R S R E Q U I R E D F O R C H R O M O S O M E S E G R E G A T I O N I N B U D D I N G Y E A S T B Y BENJAMIN CHENG B . S c , Simon Fraser University, 1998 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree o f D O C T O R O F P H I L O S O P H Y in The Faculty o f Graduate Studies (Medical Genetics) The University of British Columbia October 2005 © Benjamin Cheng, 2005 Abstract During the course of 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 of identical sister chromatids must remain tethered together until all pairs of sister centromeres have attached to the mitotic spindle in a b i -oriented manner, a state termed metaphase. Once metaphase has been successfully achieved, the initiation of anaphase can take place, and sister chromatids are pulled apart to the two daughter cells. Errors in this process lead to chromosome missegregation (chromosome loss or non-disjunction) and result in 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 in 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 of the yeast kinetochore and is not required for homologous recombination repair of D N A damage, but is important for sister chromatid cohesion. Using co-immunoprecipitation and mass spectrometry analysis, we identified a protein-protein interaction between Rad61p and Ded lp , an R N A helicase o f the D E A D 11 box family that has important roles in initiation of translation and m R N A splicing. D e d l p binds chromatin and may have direct roles in 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 in the nucleus for processes that are important for sister chromatid cohesion and for chromosome segregation. i i i TABLE OF CONTENTS Abstract... i i Table o f Contents iv Lis t o f Tables v i i i L is t o f Figures ix List o f Abbreviations x i Acknowledgements x iv CHAPTER 1. INTRODUCTION: CHROMOSOME SEGREGATION IN YEAST AND HUMANS... 1 Introduction 2 Consequences o f Aneuploidy in Humans 2 Budding yeast as a model organism 6 The Ce 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 Ce l l Cyc le . . .36 D N A Damage Checkpoint / S phase checkpoint 37 The Spindle Assembly Checkpoint 39 Chromosome Transmission Fidelity Mutants and Characterizing Novel Genes Involved in Chromosome Segregation.... 41 Overview and Scope of the Thesis 44 CHAPTER 2. Analysis oiSGTl genetic and physical interactions and identification of a factor involved in chromosome segregation, RAD61 / CTF6 46 Introduction 47 Materials and Methods 48 Results 55 Identification o f High Copy Suppressors o f sgtl-3 (Suppressors of the G2 allele of SGT1, SOGs) 55 SOG2 and SOG3 do not encode Sgt lp 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 SGT1 two hybrid interacting gene required for chromosome transmission fidelity 60 Y D R 0 1 4 W corresponds to a ctf mutant (CTF6) as wel l as a rad mutant (RAD61).... 64 rad61A mutants exhibit a cell cycle delay in 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 in sister chromatid cohesion and potentially in establishment 96 B y ChlP-chip analysis Rad61p associates with regions around centromere D N A 100 Interaction between Rad61p and Ded lp 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 Wide Two-hybrid screen ofSGTl identifies RAD61 122 Rad61p and its role in sister chromatid cohesion 124 Rad61p and Ded lp interaction 125 D e d l p and its role in chromosome segregation 126 v i R N A Helicases in Human Cancers 127 RAD61 and potential human homologues 130 Conclusion 130 References 133 vn L I S T O F 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 in the ctf mutant collection 64 Table 2-4 Double Strand Break Induction - Rad52 foci formation is unaffected in rad61A cells 77 C H A P T E R ni 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 -peptides that were isolated in multiple copies 104 Table 3-5 DED1 mutants and their sectoring phenotypes 109 v i i i L I S T O F F I G U R E S 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 of the temperature sensitivity of sgtl-3 by overexpression of SOG2 and RPS29A 57 Figure 2-2 sog2 and sog3 deletion mutants show similar sensitivity to benomyl as wi ld type strains 59 Figure 2-3 Sog2p and Sog3p do not co-IP with Sgtlp in yeast extracts 60 Figure 2-4 rad61A mutants sector 62 Figure 2-5 Co-immunoprecipitation from yeast extracts does not indicate an interaction between Rad61-13MYCp and S g t l - 6 H A p 63 Figure 2-6 rad61A / rad61A diploid cells have a G 2 / M accumulation dependent on Mad2p and are resistant to benomyl 66 Figure 2-7 Rad61-13MYCp is stable through the cell cycle with a long half-life 69 Figure 2-8 Rad61p localizes to the nucleus by indirect immunofluorescence and live cell imaging 71 Figure 2-9 A small fraction of 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 C H A P T E R 3: 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 genome wide S G A screens from Tong et al., 2004 95 Figure 3-3 rad61A strains display a cohesion defect that is l ikely a result of defective establishment 99 Figure 3-4 Rad61p by ChlP-chip analysis is specifically enriched at Centromere D N A 102 Figure 3-5 Protein-protein interactions of Rad61p and D e d l p and unconfirmed interactions of Rad61p with A l t l p and Vps3p. . . . 105 Figure 3-6 D e d l p binds chromatin but the majority of the protein is in 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 C H A P T E R 4: Figure 4-1 Potential Human Homologues o f RAD61 131 L I S T O F A B B R E V I A T I O N S A P C / C - Anaphase Promoting Complex / Cyclosome b p - base pairs C E N - centromere C L N - Chromosome Instability C M P - Chromatin Immunoprecipitation C D K - Cyc l in Dependent Kinase C F - Chromosome Fragment C F P - Cyan Fluorescent Protein C K I - C D K Inhibitor C T F - Chromosome Transmission Fidelity D M S O - Dimethylsulphoxide D S B - Double Strand Break E M S - Ethylmethanesulfonate F A C S - Fluorescence Activated Ce l l Sorting G l - Gap 1 (Growth before D N A replication phase of cell cycle) G 2 - Gap2 (Growth after D N A replication phase o f cell cycle) G A P - GTPase activating protein G E F - Guanine nucleotide Exchange Factor G F P - Green Fluorescent Protein G D P - Guanine Di-Phosphate G T P - Guanine Tri-Phosphate H R - Homologous Recombination x i I P - Immunoprecipitation L A C - Lactose L O H - Loss O f Heterozygosity M - Mitosis M b p - Mega base pairs M I N - Microsatellite Instability M M S - Methylmethanesulfonate M R X - M r e l l p / R a d 5 0 p / X r s 2 p N H E J - Non-Homologous End Joining O R C - Origin Replication Complex O R F - Open Reading Frame P C N A - Proliferating Ce l l Nuclear Antigen P J X K - phosphoinositide 3-kinase related kinases pre-RC - pre-Replicative Complex R F C - Replication Factor C R P A - Replication Protein A R S C - Remodels the Structure o f Chromatin S - Synapsis ( D N A replication phase of cell cycle) S C F - Skp lp / Cul l in / F-box protein S D L - Synthetic Dosage Lethality S L A M - Synthetic Lethal Analysis on Microarrays S P B - Spindle Pole Body s s D N A - single stranded D N A T E T - Tetracycline U B C - Ubiquit in Carrying Enzyme U T R - Untranslated Region V F P - "Venus" yellow fluorescent protein Y F P - Ye l low Fluorescent Protein Y P D - Yeast Proteome Database Y P D - Yeast Extract Peptone Dextrose x i i i Acknowledgements M a n y people were instrumental in this thesis being completed. I would like to take some time now to acknowledge and thank them. I would like to thank my supervisor, Ph i l Hieter, for his advice, encouragement and guidance during the course o f both my thesis work and the subsequent thesis writing. Phi 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. Ph i l also provided an atmosphere conducive to research by having excellent scientist that facilitated interactions and I would like to thank the members of the Hieter laboratory, past and present. I would especially like to thank Katsumi Kitagawa, who as a post-doctoral fellow in 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 of 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, Kris t in Baetz, Scott Givan, Daniel Kornitzer and V i v i e n Measday. V iv i en read a draft o f the thesis and provided valuable critical feedback. Thanks also go to members of 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 Yuen, Shay Ben-Aroya, K i r k McManus , Jan Stoepel, Ben Montpetit, and Dave Thomson (Phil 's assistant). I would also like to thank my scientific colleagues that have contributed to this work. We participated in collaborations with Gerard Cagney in Stan Fields' lab at the University o f Washington who performed the initial two hybrid screen that identified x iv Rad61p, Xuewen Pan in Jef Boeke's lab at Johns Hopkins who performed the diploid-S L A M , Mark Flory in Rudi Aebersold's lab at the University o f Washington who performed the tandem mass spectrometry analysis, and Anthony Borneman in Michael Snyder's lab at Yale University who performed the CMP-chip analysis. I would like to thank my committee members, Carolyn Brown, A n n Rose, and Miche l Roberge, who provided guidance and advice through my thesis work and encouraged me to finish and write. They also provided valuable feedback on my thesis draft. I would like to acknowledge and thank all my friends that have helped me through these years as I have been working on my thesis. I would especially like to thank people that I have played Ultimate with in 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 of the North West Diplomacy Group, Alfred Loh , John Lee, and many others. Without these two outlets the sometimes frustrating work of research would have been that much more frustrating and more difficult. I would also like to thank members of 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, my family. I would like to thank my parents, Moses and Dorcas Cheng, who have supported me unconditionally through the many years o f school. I would like to thank my siblings, May , Calvin and Joshua who encouraged me through my degree. Finally I would like to thank my wife, Angela, who during the times that I was ready to stop and run away, encouraged me and through her patience, wil led me into getting this thing done! She has xv been and continues to be a wonderful blessing in my life. I would like to dedicate this thesis to her and to our little genetics experiment growing inside her! During the course of 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. x v i CHAPTER 1: INTRODUCTION CHROMOSOME SEGREGATION IN YEAST AND HUMANS 1 Introduction The proper replication and segregation of the genetic material in dividing cells is a fundamental process in 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 of the daughter cells. Errors in this process can lead to aneuploidy, a state in which a cell does not have the proper complement of chromosomes. In aneuploid cells, one or more chromosomes are missing or there is an excess number o f chromosomes. Aneuploidy can cause the uncovering of recessive mutations, or result in cell death or adverse growth effects. Aneuploid cells and genomic instability are also important factors that contribute to human diseases such as Down 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 in 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 of 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 of loss of heterozygosity ( L O H ) thereby increasing the 2 chances o f uncovering recessive mutations (e.g. in tumour suppressor genes) or by leading to higher rates of dosage imbalances of 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 by mutations in the mismatch repair genes (e.g. hMLHl and hMSH2) and is characterized by microsatellite instability (MIN) . The M I N phenotype and hereditary pre-dispositions to colon cancer arising from mutations in the mismatch repair genes were discovered based on analysis o f the M I N phenotype in 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 in key genes and C I N by L O H and/or dosage imbalance of 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 in one o f the cells in the population, and a similar process o f selection and clonal expansion would occur. Accumulating six to ten of 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 of genes are known that could be responsible and many more are yet to be discovered. Researchers in recent years have screened for somatic mutations in candidate C I N genes implicated in genome stability based on sequence similarity to genes identified in 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 in non-CIN cell lines causes a dominant negative effect leading to chromosome instability (Cahill et al., 1998). A recent report showed that biallelic mutations in 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 in cancer initiation/progression. Another spindle assembly checkpoint protein first characterized in budding yeast, MAD2, has also been found to be transcriptionally silenced in 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 in 100 candidate genes in a panel of 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 in cancer. These mutations account for <20% of the colon tumours (Wang et al., 2004) and, therefore, the genetic basis for chromosome instability remains unknown in the majority o f colon tumours. Finding additional C I N genes in model organisms in order to sequence their human homologues as candidate genes in tumour cell lines w i l l help to identify the complete spectrum o f C I N genes 4 responsible for aneuploidy in cancer. The success in 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 in cancer. Characterizing the genes that are involved in C I N tumours can provide practical applications in 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 of one o f the spindle assembly checkpoint proteins (hMADI) can cause massive chromosome loss and non-disjunction, leading to apoptotic cell death in these aneuploid cancer cell lines (Kops et al., 2004). Knowing the underlying mutations involved in aneuploidy in 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 in budding yeast with the cancer gene mutation that is causing the aneuploidy can help identify candidate proteins in tumour cell lines that when targeted by a drug can cause death to the cancer cell without ki l l ing adjacent normal cells. In addition, understanding the C I N mutational spectrum can lead to sub-classification of tumours based on the gene mutated and this could lead to improved diagnostics, prognosis and predictions of response to different treatments (Rajagopalan and Lengauer, 2004). Finding genes that are involved in 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 in people pre-disposed to inherited forms of cancer. Many o f the factors that contribute to genomic stability in mitosis are also important during meiosis and understanding their function could lead to insights into the 5 generation o f trisomies in humans such as trisomy 21 in Down Syndrome. Non-disjunction events leading to aneuploidy in the first meiotic division in female oocytes account for the majority o f trisomies (Hassold and Sherman, 2000). Trisomies occur in 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 in the spindle assembly checkpoint or sister chromatid cohesion have been postulated to be one o f the hits required for generating trisomies. The generation of 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 of non-disjunction and is postulated to be the other hit required for generating trisomies. The combination of these two hits contributes to non-disjunction when the first meiotic division is completed shortly before ovulation (Hassold and Sherman, 2000). Investigating factors involved in chromosome segregation in mitotic cells and investigating their roles in meiosis could lead to understanding the mechanisms of 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 in the mitotic cell cycle. Components and pathways involved in 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 In haploid spores that are encased in 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 of opposite mating types can mate and form diploids, and following meiosis, hybrid spore products can be detected and analyzed with novel combinations of mutations (reviewed in Rose et al., 1990). One of 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, cel l biological, and biochemical characterization in 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 in eukaryotic cells. Morphology o f individual yeast cells can be correlated with a particular stage o f the cell cycle as shown in Figure 1. Unbudded cells are in 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 G2. 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 in the cell cycle. They helped to isolate proteins that were 7 Interphase chromatin Metaphase to anaphase transition cohesin dissociated and cohesion destroyed I Kinetochore 0 Nucleus • Cohesin - • Mitotic Spindle 0 Spindle Pole Body Chromatid 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 by homology, other eukaryotes (Hartwell, 1980; Wood and Hartwell, 1982). Dr . Leland Hartwell won the Nobel 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 of 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 in budding yeast. The experimental tractability of budding yeast facilitates the testing of the validity o f novel genomic and proteomic data sets. There are -6000 genes in budding yeast, -1100 o f which are essential for viability; this has led to the generation of 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 Mega base pairs (Mbp), and the centromere is approximately 125 base pairs (bp), whereas human chromosomes are approximately 150 M b p and centromeres in human cells can be as large as 5 M b p . (Loidl , 2003). One microtubule is bound to each yeast kinetochore whereas dozens of 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" in which the nuclear envelope does not breakdown during the course o f mitosis (Loidl , 2003), and the spindle pole bodies remain anchored in the nuclear membrane during mitosis. In contrast, mammalian cells undergo an "open mitosis" in 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 of 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 in the budding yeast cell cycle is the point in G l when the cell begins the vegetative cell cycle and cannot thereafter be affected by mating pheromone. Another example would be the metaphase to anaphase transition when sister chromatids are pulled apart. A breakdown in cell cycle control could lead to errors that contribute to chromosome missegregation and aneuploidy. Two C I N genes mutated in 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. Cyc l i n Dependent Kinases ( C D K s ) serve as the regulators of the cell cycle and, in 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 in 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 in 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 by phosphorylation and the binding o f inhibitors ( C D K Inhibitors, CKIs) such as S i c l p in budding yeast. Cdc28p and Pho85p are the C D K s involved in cell cycle progression in budding yeast with Cdc28p being the main regulator (Mendenhall and Hodge, 1998). Cdc28p is expressed and maintained at a high level throughout the cell cycle and cycl in binding t controls its activity. In budding yeast, G l cyclins (C ln lp , 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 lb lp , 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 of m R N A s and proteins during the cell cycle is controlled by phosphorylation of transcription factors by C D K s (Wittenberg and Reed, 2005). These proteins are important for events that are coordinated in 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; Ba i et al., 1996). Proteolysis of target proteins is a mechanism for cells to proceed through a "gate" in 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 in 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 by 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. Ubiquit in mediated proteolysis is well conserved in 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 E2 , a ubiquitin conjugating enzyme (or Ubiquit in Carrying Enzyme, U B C ) . A n E3 complex, ubiquitin ligase, w i l l bring substrate proteins and the UBC-ubiqui t in together and E3 complexes provide the specificity in the targeting o f proteins. 12 The Anaphase Promoting Complex / Cyclosome ( A P C / C ) and the S C F complex (Skplp / Cu l l in / F-box protein) are E3 complexes that are important for cell cycle proteolysis (for reviews see Peters, 1998; Vodermaier, 2004; Page and Hieter, 1999; Wil lems et al., 1999; Zachariae and Nasmyth, 1999). The two complexes each have a cul l in family protein (Apc2p in the A P C / C and Cdc53p in the SCF) that acts as a scaffold and structural protein aiding in E2 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 in the A P C / C and R b x l p in the S C F complex) for UBC-ubiqui t in 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 WD-40 repeats, which w i l l contribute to protein-protein binding and are responsible for bringing substrates together with the UBC-ubiqui t in . The A P C / C (Cdc20p) is responsible for targeting proteins that are important for the metaphase to anaphase transition (such as Pds lp , discussed below) and also passage through mitosis. A P C / C (Cdhlp) is responsible for proteolysis later in 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 of 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 ln lp and Cln2p) and Cdc6p, a protein involved in 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 in order to replicate the entire genome in 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 Ce l l Nuclear Antigen, P C N A ) (Bell and Dutta, 2002). In budding yeast, these origins of replication are approximately 100 to 150 base pairs and include highly conserved A elements and less wel l conserved B elements that may aid in unwinding o f the D N A double helix (Bell , 1995). The sequence of origins of 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 Ore l - 6), Cdc6p, Cd t lp 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 of 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 of the S C F (Cdc4p) and is targeted for degradation in 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 Mcm2 - 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 in 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 of Cdc6p and O R C (Hua and Newport., 1998). M c m 2 -7 is regulated also in a cell cycle manner but not by proteolysis. Phosphorylation o f subunits in the Mcm2 - 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 of replication that w i l l replicate only once in the cell cycle (Bell and Dutta, 2002). There is some evidence that Mcm2 - 7 functions as a D N A helicase but this has not been completely proven (You et al., 2002; Lee and Hurwitz, 2002). Once the pre-RC is formed at origins of replication in 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 by the pre-RC and direct the initiation o f replication by recruiting other proteins such as replication protein A (RPA) , a single stranded D N A (ssDNA) binding protein that protects and stabilizes s s D N A generated by 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 of R f c l - 5) displaces the polymerase, and acts to load Proliferating Ce 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 wel l understood. Proper timing and control of D N A replication plays an important role in genome stability. Mis-regulation of origins of 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 by 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 of 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 in sections below. 16 The Mitotic Spindle and Chromosome Segregation The mitotic spindle is the specialized structure assembled during mitosis in 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 of a hetero-dimer of a-tubulin (Tublp and Tub3p in budding yeast) and P-tubulin (Tub2p in budding yeast), two proteins that share approximately 50% identity (Desai and Mitchison, 1997). The hetero-dimers of a -tubulin and P-tubulin are arranged in 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 in human cells and spindle pole bodies in budding yeast). The head to tail interactions of the hetero-dimers are arranged into linear protofilaments that w i l l associate to form a hollow cylindrical tube of a microtubule (Kline-Smith and Walczak, 2004). Microtubules are dynamic structures and the prevailing model for their action is that of dynamic instability driven by four factors: 1) the rate of 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 of microtubules 17 during growth. During or shortly after polymerization, the G T P bound form of 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 P-tubulin 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 in budding yeast consists of 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 in an anti-parallel array (interpolar microtubules). The spindle pole body is also the site of 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 of 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 by 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 in mitotic spindle formation. Once sister chromatids have formed stable kinetochore microtubule attachments (bi-oriented to opposite spindle poles), a process that is monitored by 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 by 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 in 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 of 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 by de-polymerization (Seshan and Anion , 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 of 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 in 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 in 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 NdclOp, Ctf l3p, Cep3p and Skplp) that binds to the CDEIII region o f the centromere. A l l o f the components of 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 H3 variant that replaces H3 in 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 wel 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 of the C B F 3 complex may reflect the difference in centromere sequence between yeast and humans. Skp lp is an especially interesting protein as it plays roles in two distinct processes. Skp lp was originally identified in our laboratory as a high copy suppressor of a temperature sensitive mutant, ctf 13-30 (Connelly and Hieter, 1996), and characterized as a component of the kinetochore. Skp lp 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 Skp lp and their F-box domains; Ct f i3p is also an F-box protein and binding o f Skp lp to Ctf l3p requires Ctf l3p ' s F-box domain (Russell et al., 1999). Temperature sensitive alleles of SKP1 fall into two different classes; mutants that arrest at the restrictive temperature with a G l / S profile with a defect in S C F function, e.g. skpl-3, and mutants that arrest at the restrictive temperature with a G 2 / M profile and a defect 21 in 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 Sgt lp was found to be a protein that directly interacted with Skp lp and functioned with Skp lp at the kinetochore and the S C F . SGT1 is an essential gene and temperature sensitive alleles of SGT1 can be distinguished in 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, Sgt lp interaction with Skp lp is mediated by the HSP90 chaperones and their activity is required to activate Ct f l3p 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 sub-complexes but together they form a large macro-molecular complex at the central kinetochore that can be co-purified by biochemical assays such as co-immunoprecipitation (Pot et al., 2003; Measday et al., 2002; De 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 of 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). Many 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 wi ld 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 Kip3p (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 in a mono-polar manner to the old SPB. Kinetochore microtubule capture and release is not wel l understood but the mechanisms for regulating proper bi-polar attachment have begun to be elucidated. The current model involves a kinase complex consisting of S l i l 5p / Ip l lp that senses the lack o f tension generated by mono-polar attachment and phosphorylates D a m l p , Ndc80p and NdclOp (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 wel l as this mechanism to ensure bi-polar attachment, the spindle assembly checkpoint, which in budding yeast consists of 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 in time from chromosome segregation. After S phase each chromosome w i l l consist o f two identical sister chromatids and in 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 of cohesion can lead to precocious sister chromatid separation leading to chromosome loss or non-disjunction. The mechanism o f sister chromatid cohesion is wel l conserved in 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 of at least four subunits, a heterodimer o f two S M C (Structural Maintenance of 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 in a head to head and a tail to tail conformation with the circumference of the cohesin ring consisting o f the flexible coiled coi l regions of Smc lp and Smc3p. The tails o f the Smc lp 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 ATPase domains o f A B C transporters and it is thought that their binding is dependent on A T P binding. Scc lp binds the head region o f S m c l p and 24 cohesin coiled coil heads 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 Smc lp and Smc3p form the circumference o f the ring, with their tails forming a hinge region and their heads associating to close the ring. Scc lp binds to the head region with Scc3p associating with S m c l p and Smc3p through Scc lp . Smc3p, with Scc3p binding to Scc lp (Haering et al., 2002). Scc lp can bind both A T P and A D P bound forms o f Smc lp 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 Scc lp and Scc3p (Arumugam et al., 2003; Weitzer et al., 2003). A l l four subunits of the cohesin complex are essential for viability in budding yeast (Saccharomyces Genome Database, SGD) . 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 in budding yeast) (Ciosk et al., 2000) and this loading occurs in 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 of sister chromatid cohesion is not wel l understood but is known to require replication and the acetyltransferase activity of E c o l p / Ctf7p (Skibbens et al., 1999; Toth et al., 1999). The proteinaceous ring structure of 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 by 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 in order to remain associated with one another (Huang et al., 2005; Campbell and Cohen-F ix , 2002). Sister chromatid cohesion must be maintained through G2 and into mitosis when all sister kinetochores have achieved bipolar attachment to the mitotic spindle. When bipolar attachment occurs cohesion must be dissolved between the sister chromatids in order to allow the sister chromatids to be pulled apart. This dissolution o f sister chromatid cohesion is dependent on the cleavage of the Scc lp 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 in budding yeast, is tightly regulated by a protein called Securin, Pds lp in budding yeast, in order to ensure that Scc lp cleavage and the dissolving o f sister chromatid cohesion does not occur too early (reviewed in Yanagida, 2000). Securin is a protease inhibitor and binds to Separase to inhibit its activity. Pds lp 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 of the A P C / C (Cdc20p) induced by D N A damage or improper attachment of mitotic spindles and kinetochores can lead to high Pds lp levels that w i l l generate a wait anaphase signal. In unperturbed cell cycles, once all sister kinetochores are properly attached, Pds lp is ubiquitinated by the A P C and is degraded by the 26S proteosome. Its degradation triggers Esp lp protease activity, which w i l l then cleave Scc lp leading to sister chromatid segregation. Pds lp is also required for the nuclear localization of Esp lp and in that manner also plays a role in activating Separase (Jensen et al., 2001). S c c l ' s cleavage by Esp lp 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 Smc lp 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 in order not to interfere with cohesin in the next cell cycle (Rao et al., 2001). After mitotic exit and cytokinesis, the new daughter cells in G l w i l l begin preparing for a new cycle o f sister chromatid cohesion by forming the S m c l p / Smc3p heterodimer in anticipation of Scc lp ' s transcription and translation in late G l (Uhlmann, 2004). 27 One important difference exists between sister chromatid cohesion in budding yeast and sister chromatid cohesion in human cells. In budding yeast, the cohesin complex remains associated with centromeres and chromosome arms until anaphase whereas in 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 in 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 of sister chromatid cohesion (Stead et al. , 2003). Characterization and isolation of the proteins involved in 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. Wi th no 28 microtubules to pull sister chromatids apart, wi ld type cells should have sister chromatids in close proximity, whereas cells with defective cohesion show precocious separation of 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 in these strains and visualization o f the G F P signal is used to assess the proximity of sister chromatids containing the operator arrays. Defects in 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 in genes coding for subunits o f the cohesin complex (SCC1, SCC3, SMC1, SMC3), or in the cohesin loading complex (SCC2, SCC4), caused a high increase in the percentage o f cells that exhibit two G F P signals (up to 60% of mutant cells exhibited 2 G F P signals compared to 8% to 10% in wi ld 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 wi ld type cells). The function o f many of these proteins is unclear as they do not appear to be part o f the cohesin complex or part of the chromatin loading process. Many o f the proteins have been categorized as factors needed for establishment of 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. Ctf l8p replaces R f c l p in the R F C complex and is found in a complex with the other four subunits (Rfc2,3,4 and 5) as wel l as CtfBp and D c c l p (Mayer et al., 2001). This alternative R F C complex plays a role in the establishment of sister chromatid cohesion and deletion mutants o f CTF8, CTF18 and DCC1, as well as mutants of RFC4 show cohesion defects when tested by 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 of 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 in 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 in sister chromatid cohesion include C h l l p , a D N A helicase, M r e l l p involved in D N A damage checkpoint signaling (discussed below), Ctf4p, and Tof lp . T o f l p is a topoisomerase I interacting protein that is involved in the D N A damage response pathway in S phase. Proteins involved in 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 in sister chromatid cohesion (Baetz et al., 2004; Hsu 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 SWI/SNF family of ATP-dependent chromatin remodelers (Wang, 2003). Their main function is to reposition nucleosome arrays and change the underlying chromatin structure of chromosomes in 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 of 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 with one group claiming that R S C mutants have defective loading o f Scc lp 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 in G 2 / M . Transcription o f genes involved in sister chromatid cohesion is not down-regulated in transcriptional profiling experiments performed on R S C mutants so the effect of R S C on sister chromatid cohesion is l ikely not due to R S C 31 remodeling chromatin to allow access of transcription factors to such genes. The R S C complex may play a role in the positioning o f nucleosomes during replication and in 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 of chromosome segregation to one round of D N A replication, thereby forming In haploid progeny from a 2n diploid parent. In the first round of 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 in budding yeast (Toth et al., 2000). Rec8p, a variant o f Scc lp , replaces Scc lp 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 Esp lp during meiosis I (after Pds lp is ubiquitinated and degraded by the A P C , as in mitosis), and Rec8p containing cohesin is released from chromosome arms leading to loss of 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 of two sister chromatids) are pulled to opposite spindle poles (Buonomo et al., 2000). Rec8p is protected from cleavage at the kinetochore by Sgolp 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). Sgolp 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 in mitotic kinetochore and checkpoint functions sensing tension (Watanabe and Kitajima, 2005; Indjeian et al., 2005). Sgolp ' 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 of D N A damage is of fundamental importance to genome stability. Double strand breaks (DSB) are particularly dangerous as they cause a disruption to both strands of D N A (Aylon 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 in 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 of D S B s , non-homologous end joining (NHEJ) and homologous recombination (HR) (Aylon and Kupiec, 2004). H R does not result in the loss of genetic material (i.e. D N A sequences surrounding the D S B ) and is the primary pathway of repair o f D S B s in 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, in 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 in 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 post-replicative repair or i f there was some other direct function of 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 (Smcl) is phosphorylated by A T M (Tel l ) in response to D N A damage induced by ionizing radiation (Kitagawa et al., 2004; K i m et al., 2002b; Yazd i et al., 2002). This phosphorylation is necessary for the checkpoint response. Recent work in 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 DSBs . Through a series of 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 in budding yeast the cohesin complex is recruited to flanking regions of 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 of Histone H 2 A X (the major Histone H 2 A variant in budding yeast) at sites of 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 in the D N A damage checkpoint response pathway and recognition of 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 of D S B . M r e l l p is in a complex (Mre l Ip/Rad50p/Xrs2p, M R X complex) that localizes to D S B s and may serve as the early sensor and indicator of D N A damage and Rad53p is a kinase involved in 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 in H R repair (Sjogren and Nasmyth, 2001; Strom et al., 2004; Unal et al., 2004). Are there other functions that cohesin is playing at sites of D S B s and D N A damage? In human cells the two S M C subunits of 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 in the cohesin complex (Lehmann, 2005). Many o f the deletion mutants o f the non-essential genes coding for factors involved in 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 in budding yeast, their sensitivity to D N A damaging agents could represent their action in dealing with D N A damage. On 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 of 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 in the cell cycle occur in 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 of 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 wel l conserved from budding yeast to humans and the medical importance o f many o f the proteins involved in these checkpoint functions is demonstrated by the mutations in genes encoding these proteins that have been found in 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 of D N A damage it is generally assumed that processing of the D N A damage into specific forms is a part of 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 Rpa 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 Radl7p /Ddclp /Mec3p onto sites o f D N A damage (Majka and Burgers, 2003). Al so , the M R X complex localizes to D S B s and acts as a signal at D S B s (D'Amours and Jackson, 2002). These proteins are all involved in signaling D N A damage and are upstream o f proteins involved in 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 in response to the initial D N A damage signal. The two chief mediators are phosphoinositide 3-kinase related kinases (PIKKs) , M e c l p ( A T R in 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 by M e c l p and T e l l p include C h k l p and Rad53p, both of which are important for inducing cell cycle arrest (Longhese et al., 2003). D N A damage at various stages of the cell cycle can cause a halt in cell cycle progression until there has been time to repair the damage. G1- , S- and G2-arrest can all occur in response to D N A damage. The G l arrest in response to D N A damage in budding yeast is weak and lasts for approximately an hour. The response is mediated by Rad53p phosphorylation of Swi4p/6p transcription factors thereby inhibiting and delaying the production of 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 l ikely reason for this weaker G l D N A damage response is that homologous recombination from sister chromatid templates is the major repair mechanism in budding yeast (Nyberg et al., 2002) . D N A damage that is encountered in S phase, such as D N A adducts caused by 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 in S phase (dependent on many o f the factors involved in 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, Pds lp , and preventing the release o f E s p l p and the subsequent degradation and dissociation of 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 in G2 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) The Spindle Assembly Checkpoint 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 by 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 in G 2 / M by preventing the metaphase to anaphase transition. Many of the proteins involved in the 39 spindle assembly checkpoint were first isolated in 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 Lew and Burke, 2003; Skibbens and Hieter, 1998; Amon, 1999). The signal that the spindle assembly checkpoint responds to has been proposed to be either lack of tension exerted at kinetochores or the lack of 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, Ip l lp may destabilize kinetochore microtubule attachments that lack tension and signal this lack o f tension. Alternatively, the transient unattached kinetochores that have been caused by Ip l lp activity may be the signal (Tanaka, 2005). Both processes are l ikely important as cellular localization of 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 in the spindle assembly checkpoint have been extensively characterized in 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; Goh and Kilmart in, 1993) and mutations in members of 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 in mutants o f the N D C c 8 0 complex and this complex may play a role in recruiting and regulating the proteins involved in the checkpoint (Janke et al., 2001; McCleland 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 in S P B duplication and a protein kinase) are proteins important for the spindle assembly checkpoint (Lew and Burke, 2003; Amon, 1999). The prevailing hypothesis on how the checkpoint induces cell cycle arrest is that in the event of kinetochore microtubule defects, Mad2p is exchanged from a Madlp /Mad2p complex to a Cdc20p/Mad2p complex. Cdc20p is one of the adaptors for the A P C / C ( A P C / C (Cdc20p) is responsible for the ubiquitination and degradation of Pds lp securin) and Mad2p binding o f Cdc20p prevents this activity, thus maintaining high levels o f Pds lp and preventing the release o f E s p l p , thus preventing the degradation and dissociation of 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 in chromosome segregation, different screens have been performed in 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 in 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 of sectoring (red sectors in a white yeast colony) and was the basis for isolating mutants that increased the rates of sectoring (and by extension, the rate o f loss and non-disjunction o f endogenous chromosomes) compared to wi ld type yeast cells. The initial set of 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 in kinetochore function. To identify the relevant mutants, secondary assays were developed. One example of a secondary assay used is the centromere transcriptional readthrough assay assessing kinetochore proteins binding to C E N in 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 wi ld type strains did not allow expression (Doheny et al., 1993). Another example is Synthetic Dosage Lethality (SDL) whereby overexpressing a gene involved in kinetochore function may have no effect in wi ld type cells but may cause lethality in a kinetochore defective mutant (Krol l et al., 1996; Hyland et al., 1999). 42 The lethality is most likely due to compromising what few proper kinetochores have assembled by further upsetting the stochiometric balance in the cell. Using these secondary assays, several ctf genes have been identified and subsequently characterized as members of the inner and central kinetochore (such as Ctf l3p, Ctf l4p (NdclOp), Ct f l9p , C tOp , 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. High 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). High copy suppression screens have been used to identify interacting proteins as wel l as proteins involved in 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 of the S C F complex (Bai et al., 1996). SGT1 (Suppressor o f the G2 allele of SKP1) was found as a high copy suppressor of 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 of 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 Overview and 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 in the number o f proteins that have been identified and placed at the kinetochore. Many o f the proteins that have been placed in 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. Work in the yeast community in the last few years has also elucidated much o f the mechanism o f sister chromatid cohesion and how it affects bi-orientation of 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. We began this work using SGT1 as a starting point. A class of temperature sensitive mutations in SGT1 display defects in kinetochore function and we were interested in identifying high copy suppressors o f the temperature sensitivity. We reasoned that this may identify novel components or regulators o f the yeast kinetochore. To identify potential protein partners of Sgt lp, we carried out a two-hybrid screen using SGT1 as the "bait" against the genome wide collection of ORFs in collaboration with Stan Fields. Phenotypic analysis o f deletion mutants corresponding to genes that were identified in the two screens focused our interest on an uncharacterized O R F , Y D R 0 1 4 W . M u c h of the work in the second half o f Chapter 2 and in the majority of Chapter 3 is focused on characterizing the role of 44 Y D R 0 1 4 W (RAD61) in chromosome segregation and the function of a Rad61p protein interaction partner, Ded lp . 45 Chapter 2: Analysis oiSGTl genetic and physical interactions and identification of a factor involved in chromosome segregation, RAD61 / CTF6 46 Introduction Two approaches were undertaken to identify genes that could be important for chromosome segregation and in particular, kinetochore function. Both approaches attempted to identify proteins that were playing a role at the budding yeast kinetochore in concert with Sgt lp. The first approach utilized strains that contained the sgtl-3 mutation that causes arrest in G 2 / M at non-permissive temperature. We looked for genes that when present in multiple copies, could suppress the temperature sensitive phenotype o f sgtl-3 mutant strains. This high copy suppression screen identified four ORFs , that when present in multiple copies, rescued the lethality of sgtl-3 strains at 37°C. The second approach was to identify protein partners o f Sgtlp by using SGT1 as the "bait" in a genome wide two-hybrid screen in collaboration with Stan Fields. We 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 in the second half o f the chapter were designed to characterize Rad61p's role in chromosome segregation. 47 Materials and Methods Yeast strains, growth conditions and media Yeast strains used in this study are listed in Table 2-1. Strains were grown at 25°C unless otherwise indicated. Temperature sensitivity and lethality was assessed at 37°C. Media for growth and sporulation were described previously (Rose et al., 1990). To visualize the loss of the non-essential chromosome fragment (CF), the strains were first grown in SC media lacking either uracil or tryptophan (selecting for the CF) , then either plated (200 to 300 cells per plate) or streaked to single colonies onto SC complete media with 65 fiM adenine concentration. Colonies were visualized after growth at 25°C or the temperature indicated for 3 to 4 days followed by 1 or 2 days at 4°C to increase the resolution of 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 in dimethylsulphoxide ( D M S O ) at 10 mg/mL and added to the indicated concentrations to Y P D plates, with addition of D M S O lacking drug as a control. Methlymethanesulfonate ( M M S ) in liquid form (99% pure, from Sigma) or bleomycin (10 Units /mL dissolved in 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 25°C for 3 days before replica plating to 37°C. We identified -300 yeast colonies that grew at 37°C from 4 x 10 5 yeast transformants and pooled them into 6 fractions of approximately 50 colonies each. We purified plasmid D N A from the pooled colonies (containing plasmid D N A that rescued the conditional lethality at 37°C of 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. We found 18% of 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. We found four transformants that were able to suppress the conditional lethality o f sgtl-3 at 37°C. Sub-cloning using restriction sites present in the genomic fragments was used to identify the ORFs that were responsible for suppression. Putative ORFs 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 of the O R F . 49 Co-immunoprecipitation experiments from yeast extracts Co-immunoprecipitations were performed as described previously in Measday et al., (2002). In brief, yeast extracts were generated using glass bead lysis, equal amounts of 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 in extract buffer for a minimum of 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 by Uetz et al., (2000). Putative two hybrid positives were retested and confirmed by 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 in Hyland et al., (1999). Chromosome Spreads and Chromat in Binding Assays Chromosome spreads were performed as previously described in 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 Rad61-13MYCp staining. D A P I was used as the marker for chromatin D N A . Chromatin Binding was performed as previously 50 described in Donovan et al., (1997) with histone H4 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 of 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 anti-Cdc28 antibodies and the 9E10 A n t i - M Y C antibody from Covance at the time points indicated as wel l as preparing F A C S samples. For assays investigating the half life of Rad61p, we added cyclohexamide to a concentration of 10 pg/mL and protein extracts were made at the time points indicated. For testing the stability of Rad61p in sgtl-5 mutants compared to wi ld type, we cloned HA-Rad61 into a G A L expression vector and transformed it into Y K K 57 (sgtl-5) and wi ld type strains. Cultures were grown in l iquid media selecting for the plasmid vector with glucose as the carbon source. Cells were subsequently harvested, washed and allowed to grow in raffinose for 2 hours. Galactose was added to a concentration of 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 in F P M (Synthetic Complete medium supplemented with adenine and 6.5 g/L sodium citrate) in order to reduce auto-fluorescence. Unless otherwise indicated microscope pictures are o f live cells harvested 51 during log growth, and resuspended in 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 by addition of 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 Axioplan 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 in the figure legends. Indirect immunofluorescence was performed as previously described in 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 of 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 wi ld 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 of the H O endonuclease from the G A L - H O plasmid in 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 in Lisby et al., (2003). 52 Table 2-1 List of yeast strains used in this chapter Strain Genotype Reference Y P H 4 9 9 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 Sikorski and Hieter., 1989 Y P H 5 0 0 Mztaura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 Sikorski and Hieter., 1989 YPH501 Mata /a ura3-52/ura3-52 Iys2-801/lys2-801 ade2-101/ade2- Sikorski and 101 Hieter., 1989 Y B C 1 5 his3A200/his3A200 leu2Al/leu2Al trplA63/trplA63 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 This study Y B C 1 6 sog2A::HIS3 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 This study Y B C 1 7 sog2A::HIS3 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 This study Y K K 5 6 0 sog2A::HIS3 CFIII (CEN3.L.) TRP1 SUP11 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 K . Kitagawa Y K K 5 5 6 sog3A::HIS3 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 K . Kitagawa Y K K 5 2 9 sog3A::HIS3 CFIII (CEN3.L) TRP1 SUP11 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 K . Kitagawa Y B C 1 0 8 sgtlA::HIS3 6HA-SGT1-LEU2 @ Ch.ni M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 This study Y B C 1 0 9 SOG2-13MYC-TRP1 sgtlA::HIS3 6HA-SGT1-LEU2 @ Ch.III M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 This study Y K K 5 7 SOG3-13MYC-TRP1 sgtlA::HIS3 6HA-SGT1-LEU2 @ Ch.III K . Kitagawa M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 Y K K 1 9 0 sgtl-5:LEU2 K . Kitagawa M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 Y C T F 7 sgtl-3:LEU2 Spencer et al., M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 1990 Y C T F 4 0 CFIII (CEN3.L.) URA3 SUP 11 ctf6-7 Spencer et al., M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 1990 Y C T F 6 0 CFIII (CEN3.L.) URA3 SUP 11 ctf6-40 Spencer et al., M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 1990 Y C T F 1 3 4 CFIII (CEN3.L.) URA3 SUP 11 ctf6-60 Spencer et al., Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 1990 YPH311 CFIII (CEN3.L.) URA3 SUP 11 ctf6-134 Spencer et al., Mata ade2-101 his3A200 ura3-52 tubl-1 1990 Y P H 3 1 2 Spencer et al., M a t a ade2-l01 ura3-52 tub2-104 1990 Y B C 1 9 7 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 This Study Y B C 1 9 8 RAD61-13MYC-HIS3 This study Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 RAD61-13MYC-HIS3 53 Table 2-1 List of yeast strains used in this chapter, continued Y B C 2 0 4 Y B C 2 0 5 Y B C 2 0 7 Y B C 3 0 9 Y B C 2 0 0 Y B C 2 0 1 YPH1555 Y B C 6 7 1 Y B C 6 7 2 Y B C 6 7 3 Y B C 6 7 4 Y B C 6 5 8 Y B C 6 6 1 Y B C 6 7 5 YPH1474 Y P H 1 0 2 0 Mata 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 Mata/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 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 sgtlA::HIS3 6HA-SGT1-LEU2 @ Ch.IIIRAD61 -13MYC-TRP1 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 sgtlA::HIS3 6HA-SGT1-LEU2 @ Ch.III RAD61-13MYC-TRP1 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 NDC10-13MYC-kanMX6 Mata 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 Mata 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 Mata ade2-l barl::LEU2 trpl-1 LYS2 RAD5 RAD52-CFP ura3::3xURA3-tetOxll21-Scel(ura3-1) his3-l 1,15::YFP-Lacl-his3-x leu2-3,112::LacO-LEU2 HO-iYCL018W(leu2-3,l 12) TetR-RFP(iYGL119W) (W303 background) Mata ade2-l barl::LEU2 trpl-1 LYS2 RAD5 RAD52-CFP ura3::3xURA3-tetOxll21-Scel(ura3-1) his3-ll,15::YFP-Lad-his3-x leu2-3,112::LacO-LEU2 HO-iYCL018W(leu2-3,l 12) TetR-RFP(iYGL119W) rad61A::HIS3 (W303 background) Mata his3Al leu2A0 metl5A0 ura3A0 rad52A::kanMX6 Mata 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 This study This study This study This study This study This study Pot el al., 2003 This study This study This study This study Lisby et al., 2003 This study Giaever et al., 2002 Mayer et al., 2001 Spencer et al., 1990 54 Results Identification of High Copy Suppressors of sgtl-3 (Suppressors of the G2 allele of SGT1, SOGs) Sgtlp is a protein that, together with its direct binding partner Skp lp , is essential for two distinct functions: 1) in the assembly of the budding yeast kinetochore, 2) and as a subunit of 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 Skplp /Sg t lp complex, temperature sensitive mutants carrying mutations in 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 in S C F function while the G 2 / M alleles are defective in kinetochore function. High 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 multi-copy suppressor screen o f an sgtl-3 mutant. A genomic library carried in 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 of 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 ORPs 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). We provisionally named Y L R 0 7 3 C and Y D R 2 6 6 C SOG2 and SOG3 for Suppressors of the G2 allele o f SGT1. To confirm that expression of these ORFs 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 in each case the suppression of the temperature sensitivity depended on the expression o f the ORFs . A n example suppression of the temperature sensitivity phenotype o f sgtl-3 by SOG2 or RPS29A, as well as a negative control, Y L R 3 8 7 C , and vector alone, is shown in Figure 2-1. Expression of Sog2p and Rps29ap in sgtl-3 strains rescued the temperature sensitivity phenotype of the sgtl-3 strain and allowed growth at 35°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 in budding yeast and 67% identical to human RPS29) and its function is well characterized in m R N A binding and translation. UFD2 is a gene involved in assembly o f multiubiquitin chains on ubiquitin-protein conjugates. The deletion mutants of RPS29A and UFD2 are viable and do not have chromosome missegregation phenotypes as measured by three different screens, the sectoring assay (loss of 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 of 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 ORFs that coded for proteins of unknown function. We performed phenotypic assays on the deletion mutants of SOG2 and SOG3 in order to investigate any potential role of Sog2p or Sog3p in chromosome segregation. Neither sog2A nor sog3A strains exhibited increased rates o f missegregation o f a marker chromosome fragment relative to wi ld type (data not shown). The strains also did not show sensitivity to the microtubule depolymerizing drug benomyl (Figure 2-2). Benomyl sensitivity is a hallmark of genes that are involved in kinetochore or sister chromatid cohesion function. In addition, SOG2 and SOG3 did not display genetic interactions such as S D L with components of the inner and central kinetochore. For example, when we overexpressed CTF13, NDC10, SKP1, SGT1, and CTF19 from galactose inducible promoters in sog2A and sog3A strains, there was no effect on growth o f the deletion mutants, as compared to wi ld type strains (data not shown). We performed co-immunoprecipitation experiments using yeast extracts to determine i f either Sog2p or Sog3p interacted physically with Sgt lp. Epitope tagged constructs of Sog2p, Sog3p and Sgtlp were made at their endogenous loci . Yeast extracts were prepared from strains containing SOG2-13MYC and SGT1-HA (lanes 1 to 3 in Figure 2-3a and 2-3b), SOG3-13MYC and SGT1-6HA (lanes 4 to 6 in Figure 2-3a and 2-3b), and SGT1-6HA alone as a negative control (lanes 7 to 9 in 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 Sog2-13MYCp or Sog3-13MYCp (Figure 2-3b, see lanes 3 and 6). From this co-immunoprecipitation experiment it appears that neither Sog2p nor Sog3p physically interact with Sgtlp. 58 sog2A sog3A Wild Type tub1-1 tub2-104 sog2A sog3A Wild Type tub1-1 tub2-104 YBC17 • • • •ft YKK560 • •• # * YPH499 * ft YPH311 • • ft YPH312 B YBC17 [ 1 1 1 YKK560 ft> YPH499 YPH311 # • • YPH312 DMSO 10/yg/mL benomyl Wihd Type ^ ^ ^ ^ ^ ^ ^ YPH499 15/vg/mL benomyl 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 in whole cell extracts, we determined that Sog2p and Sog3p were not important for chromosome segregation and do not interact with Sgt lp . We therefore did not pursue further analysis of the suppressor genes. 59 Figure 3 Sog2p and Sog3p do not co-IP with Sgtlp in yeast extracts a) Ant i -MYC IP, Ant i -MYC Western Y B C 1 0 8 YBC109 YKK529 SOG2-13MYC SOG3-13MYC SGT1-6HA SGT1-6HA SGT1-6HA tot sup IP tot sup IP tot sup IP 1 2 3 4 5 6 7 8 9 b)Ant i -HA Western Y B C 1 0 8 YBC109 YKK529 SOG2-13MYC SOG3-13MYC SGT1-6HA SGT1-6HA SGT1-6HA tot sup IP tot sup IP tot sup IP 1 2 3 4 5 6 7 8 9 Figure 2-3 - Sog2p and Sog3p do not co-IP with Sgtlp in yeast extracts. Yeast extracts from strains expressing S O G 2 - 1 3 M Y C p and Sog3-i3MYCP 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 of IP sample to detect any interaction thus the large amounts seen in the IP lanes (3 and 6). B) A n t i - H A (12CA5) Sgti-6HAp 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 Sg t l -6 H A p to the A n t i - M Y C beads. 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 in 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 of 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 of 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 wi ld type (Figure 2-4). We 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 wi ld type strains. Both 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 wi ld type or rad61A strains that contained half-sectors (or more) of red pigment in order to quantify the chromosome missegregation rate of rad61A strains and found that rad61A strains had a ~ 20x elevated rate of chromosome missegregation compared to the wi ld 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 Sgtlp and Rad61p ( Y D R 0 1 4 W , see below). We used strains expressing only SGT1-6HA (lanes 1 to 3 in Figure 2-5a, and lanes 1 and 2 in Figure 2-5b) or RAD61-13MYC (lanes 4 to 6 in Figure 2-5a, and lanes 3 and 4 in 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. We could not detect an interaction between the two proteins (see lanes 6 and 8 in Figure 2-5b) 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 YBC200 YBC201 Rad61-13MYC Sgt- Rad61- Sgt1-6HA 6 HA 13MYC #1 #2 T S I P T S IP T S IP T S IP Rad61-13MYCp 14 5 6 7 8 9 10 11 12 b) Anti-HA Western YKK529 YBC197 YBC200 YBC201 Rad61-13MYC Sgt1- Rad61- Sgt1-6HA 6HA 13MYC #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 Sg t l -6HAp (lanes 1 to 3), Rad61-1 3 M Y C p (lanes 4 to 6), and two strains expressing S g t l - 6 H A p and Rad61-13MYCp (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 Rad61-13MYCp, 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 Yuen, 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 of four independent isolates within the CTF6 complementation group. Single point mutations were identified in the coding region of Y D R 0 1 4 W in each case. The mutations were nonsense mutations that produced truncated protein products that were therefore l ikely non-functional (Table 2-3). The mutations fell into two classes; ctf6-7 and ctf6-60 were identical and ctf6-40 and ctf6-134 were identical, suggesting that the pairs of 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 G1227A, T R P to STOP) mutant diploids exhibited chromosome III missegregation at a rate lOOx higher than wi ld type strains (Spencer et al., 1990). We 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 in 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 wel l as M M S (a methylating agent), phenotypes that we were able to confirm in 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 wi ld 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 G2 cells in logarithmically growing cell populations as measured by F A C S , similar to w i ld 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 G2 accumulation (Figure 2-6b, left hand panels). When we investigated cellular morphology o f rad61A /rad61A compared to wi ld type diploids, we found a higher percentage o f cells in a logarithmically growing population as short spindle pre-anaphase cells (29% in rad61A / rad61A versus 13% in wi ld type diploid cells). The G2 accumulation was 65 dependent on Mad2p (Figure 2-6b, right hand panels). We also found that rad61A / rad61A strains were resistant to intermediate levels o f benomyl, compared to wi ld 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 b> G1 G2 G1 G2 Wild type I YPD md61A Wild type • • • • * YPD 2.5 mU/mL bleomycin 0.01% MMS 5.0 mU/mL bleomycin 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 (YBC204) and wi ld type (YPH499) 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 of logarithmically growing wi ld type diploid (YPH501), rad61A/rad61A (YBC207) , and rad61A mad2A/rad61A mad2A (YBC309) strains. G l and G2 peaks are labeled. C) lOx serial dilutions of wi ld type diploid (YPH501), rad61A/rad61A (YBC207) , tubl-1 (YPH311), and tub2 mutant (YPH312) 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. Wild type diploid rad61A/rad61A - U J Lai rad61b/rad61A tub1-1 (sensitive) tub2-104 (m.) Wild Type Diploid rad61A/rad61& tub1-1 (sensitive) lub2-104 Ires.) Wild Type Diploid rad61&/rad61A tub1-1 (sensitive) Iub2-104 (res.) Wild Type Diploid rad6U/rad61A c ,:• -0 O O © tub1-1 (sensitive) tub2-W4 (res.) Wild Type Diploid rad61A /rad61A rad61Lmad2L/ rad61& madlL 0 ug/mL benomyl 5 ug/mL benomyl 10 ug/mL benomyl 15ug/mL benomyl 66 G2 accumulation that is dependent on the spindle assembly checkpoint (Hyland et al., 1999; Mayer et al., 2001); Rad61p could potentially function in 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 in 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 de-polymerized 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 of Rad61p by quantifying the levels of Rad61p at various stages in the cell cycle. Previous experiments by others indicated that RAD61 m R N A levels are stable through the cell cycle (Saccharomyces Genome Database, SGD) . We synchronized yeast cell cultures with the addition o f a-factor mating pheromone (see Materials and Methods) causing a G l arrest. We 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 by 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 Rad61-13MYCp fusion was functional as determined by complementation of a rad61A mutation using the sectoring assay (data not shown). The constant level of the Rad61 protein could be due to a long half life o f the protein or to constant expression of Rad61p with a short half life. We investigated the two possibilities by treating cells with cyclohexamide (a translation inhibitor). After treatment with cyclohexamide we prepared yeast extracts every 15 minutes. We found that Rad61-13MYCp appeared stable and had a half life much greater than that o f Clb2p, a mitotic cyclin targeted for degradation by the A P C / C (Anaphase Promoting Complex) (Figure 2-7b) with a high turnover rate. We concluded that Rad61p is a stable protein with a long half life. We also tested to see i f overexpressed Rad61p could be a substrate for the S C F complex. We cloned an HA-Rad61 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 ld type and sgtl-5 strains. Upon induction with galactose and subsequent repression o f expression with glucose, we did not see stabilization of HA-Rad61p in the S C F mutant strains (Figure 2-7c) compared to wi ld type indicating that turnover o f excess Rad61p was not dependent on the S C F complex. We conclude that Rad61p is not a substrate of 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 (Rad61-13MYCp). In logarithmically growing cell cultures that were then fixed, Rad61-1 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 ive cell imaging of 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) RAD61-13MYCp| Cdc28p alpha-factor arrest and release G 1 G 2 LOO [ j j j I O CO CJl —J CO —k —k —k Q O O O O - k O O C J l CO o o o a-factor a-F 30 a -F 50 a-F 70 G1 G2 IE109 a - F 90 nz i ° - M i ° a-F 150 b) Cyclohexamide treatment of log cultures c) Wild type sgt1-5 R G 30 60 90 R G 30 60 90 Minutes after cyclohexamide treatment 0 15 30 45 60 75 - HA-RAD61p CDC28p 69 Figure 2-7 - Rad61-13MYCp is stable through the cell cycle with a long half-life. A ) Ce l l cultures from Y B C 1 9 8 were arrested in alpha-factor for 2 hours at 30°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 OD280 readings, and Cdc28p was used as a loading control. B) 10 />ig/mL of cyclohexamide was added (YBC198) to log phase cells (OD600 of 0.6) and protein samples were extracted at the time points indicated. Protein sample concentrations were assessed with OD280 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 wi ld type cells (YPH499) and sgtl-5 ( Y K K 5 7 ) cells. L iquid cultures were grown in selective media to maintain the plasmid overnight. Equal amounts of cells (by absorbance readings at OD600) were resuspended in 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 by 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 of the spindle pole bodies (Measday et al., 2002; Pot et al., 2003). B y these criteria, Rad61-VFP did not display characteristic kinetochore localization, but instead showed a diffuse nuclear staining at all stages o f the cell cycle in -150 cells examined (Figure 2-8b and 2-8c). We used Nic96-CFP to visualize the nuclear membrane and showed that Rad61-VFP was contained within the nucleus. 70 b) c) Anti-MYC DIC NIC96-CFP - red RAD61-VFP- green Untagged Control DAPI Merged Merged NIC96-CFP - red RAD61-VFP-green DIC Figure 2-8 - Rad61p localizes to the nucleus by indirect immunofluorescence and live cell imaging. A ) Indirect Immunofluorescence o f R A D 6 1 -1 3 M Y C cells (YBC198) . D A P I appears blue and M Y C appears red. -150 cells were examined at different stages o f the cell cycle, the figure is a representative image. B) Fluorescence microscopy on live cells (YBC673) . These images are flattened Z-stacks (11 planes at 0.2 fim per plane). Nic96-C F P appears red and Rad61-VFP appears green. -150 cells were examined, the figure is a representative image. C) Increased magnification o f live cell imaging o f Rad61-VFP Nic96-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 of Rad61p involved any binding of chromatin. To address this issue, two assays were employed. The first assay utilized the technique of 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, Rad61-13MYCp signal was found to co localize with D A P I in > 100 nuclei examined and by this assay can bind to chromatin (Figure 2-9a, left panel). Ctf8-1 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 in the high speed centrifugation step. The amount of Rad61p in equal volumes of the total mixture, the supernatant mixture (containing the soluble fraction of the protein), and the pellet mixture (containing the chromatin bound fraction of the protein) can then be assessed by western blot analysis. We used an antibody to acetylated H4 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 Rad61-VFP was isolated in the chromatin fraction while a larger fraction o f the protein was contained in the soluble fraction (Figure 2-9b). We obtained similar results using Rad61-13MYCp at different stages o f the cell cycle (data not shown). B y two different assays we found that a sub-fraction o f Rad61p in the nucleus was binding to chromatin. It therefore appears that Rad61p is involved directly in an aspect of chromosome biology necessary for chromosome segregation. We addressed the question of the chromosomal locations o f Rad61p binding in Chapter 3 using Chromatin Immunoprecipitation ( C M P experiments). 72 a) RAD61-13MYCp - in Red CTF8-13MYCp - in Red DAPI - blue DAPI - blue Rad61-VFP Untagged T S C P T S C P Figure 2-9 - A sub-fraction of Rad61p binds chromatin. A ) Chromosome spreads on Rad61-1 3 M Y C strain (YBC198) and Ctf8-1 3 M Y C strain (YPH1474) . 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 in 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 Rad61-VFP (YBC671)or a control untagged strain (YPH499) . Acetylated Histone H4 is used as a control for binding to chromatin and Carboxypeptidase Y ( C P Y ) is used as a control cytoplasmic protein. RAD61 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 by 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. We 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 in homothallic strains. The H O endonuclease induces a double strand break at a specific site in 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) in wi ld type, rad61A, and rad52A (involved in homologous recombination) strains, the rad61A and wi ld 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 by M M S treatment and the generation o f double strand breaks. We investigated the localization o f Rad61-VFP fusion protein in 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 of double strand break for repair. RAD52 is part of a large epistasis group that functions in homologous recombination. Upon induction of 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. We tested whether deleting RAD61 could affect the formation and localization of R A D 5 2 - C F P foci in response to the generation o f double strand breaks. We used strains developed in 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. We transformed in a vector that contained the H O endonuclease under the control of a galactose inducible promoter (pJH 132). W e induced double strand breaks by inducing expression o f the H O endonuclease by addition of 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). We 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 2-10b 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; wi ld 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 Rad52-CFP 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 (YPH499), rad61A (YBC204) , and rad52A (YBC675) cells were transformed with a G A L - H O plasmid. Ce 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 by the L A C operator sequence (YBC658) , 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 (YBC661) . 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 of 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 in 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), GAL-HO 346 98 51 Wild type (YPH658), GAL-HO as % 28.3% 52% rad61A (YBC661), GAL-HO 209 65 35 rad61A (YBC661), GAL-HO as% 31.1% 53.8% 77 Discussion The rationale for finding proteins that genetically or physically interact with Sgt lp at the budding yeast kinetochore was to identify novel proteins that played structural or regulatory roles important for kinetochore function. High 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 Sgtlp function, but we were unable to find proteins that were clearly linked to chromosome segregation to study. Neither o f the deletion mutants of the uncharacterized ORFs that suppressed the sgtl-3 mutant missegregated a chromosome fragment and neither o f the deletion mutants in the two ORFs harboured phenotypes indicative o f a potential role in chromosome segregation. The SOG2 and SOG3 O R F reports in the Yeast Proteome Database indicate that the functions of 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 of the S C F but found no effect upon overexpression of either protein (data not shown). Using 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 of Sgtlp indicated that the screen was working but the fact that Rad61p could not be immunoprecipitated in a complex with Sgt lp 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 in chromosome segregation. The epitope tagged 78 constructs o f the two proteins may also interfere with their physical interaction. We addressed the question o f potential Rad61p protein partners in Chapter 3 using mass spectrometry approaches. Rad61p has all the characteristics of a protein that is involved in 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 in the original ctf mutant collection. Furthermore, the diploid null mutant displays a spindle checkpoint dependent cell cycle accumulation in G 2 / M . We did not see this phenotype in the haploid null mutant. Diplo id null mutants displaying a defect (e.g. cell cycle progression delay) that is not seen in 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 in 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. Benomyl resistance has been demonstrated in deletion mutants o f genes involved in microtubule dynamics such as viklA (Manning et al., 1999) or kip3A (Cottingham and Hoyt, 1997). Kip3p is a kinesin-related microtubule motor protein and V i k l p is a protein partner o f Kar3p, another kinesin-related microtubule motor protein. Microtubules in kip3A mutant cells are longer and more stable compared to microtubules in wi ld type cells (Cottingham and Hoyt, 1997). We examined microtubules in rad61A I rad61A diploid cells but found no difference compared to wi ld type diploid cells (data not shown). The resistance to 79 benomyl o f the rad61A / rad61A strain could be due to the delay in progression through G 2 / M allowing cells time to stabilize microtubules and the mitotic spindle. We tested for localization of 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 of 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 in our strain background. We followed up on this data by looking at aspects o f homologous recombination repair o f D N A but also found that Rad61p had no detectable defect in Rad52p mediated homologous recombination repair. We also did not detect differences in Rad61p localization in 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 in 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. We decided to explore different genomic and proteomic approaches in order to understand the function o f Rad61p in chromosome segregation. The reagents in 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 in the same pathway (Tong et al., 2004; Pan et al., 2004). We 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. We 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 in the cohesion assays were in the W303 background. Yeast strains used in Chapter 3 are listed in 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 in 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 Xuewen Pan in 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). We tested for growth o f the haploids on G418 plates (200 / ig/mL in Y P D ) to select for query deletion strains, c l o n N A T plates (1 /xg/mL in 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 Two 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 of the data was as described in Tong et al., (2004). Clustering was performed by Cluster 3.0 and visualized with Treeview 1.0.8. Cohesion Assay Cohesion assays were performed as described in Mayer et al., (2001). Cells were counted using a Zeiss Axioplan 2 microscope using the lOOx objective lens. ChlP-chip Assay ChlP-chip analysis was performed as previously described in 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 in parallel, labeled and hybridized onto the same D N A chip. ChlP-chip analysis was performed by Anthony Borneman in Michael Snyder's laboratory at Yale 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 in 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 Vac Plus and tandem Mass Spectrometry was performed. Tandem mass spectrometry and analysis was performed as described in Lee et al., (2004) by Mark Flory in Rudi Aebersold's laboratory at the University o f Washington. For 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. pGEX-Rad61 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 in the soluble fraction in 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-PBS) and subsequently incubated with yeast lysate overnight at 4°C. After washing 4 times with T -PBS , 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 in Measday et al., (2002) using equal amounts o f protein. 85 Methods f rom Chapter 2 We performed chromosome spreads, chromatin binding, fluorescence and indirect immunofluorescence microscopy, and F A C S analysis of cells as described in Chapter 2 Materials and Methods. 86 Table 3-1 List of yeast strains used in this chapter Y P H 4 9 9 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 Y P H 5 0 0 Y B C 2 8 5 Y B C 2 8 9 Y B C 2 5 5 Y B C 3 2 9 Y B C 4 0 0 Y B C 4 2 3 Y B C 4 3 9 Y B C 6 7 6 Y B C 6 9 8 Y B C 6 9 9 Y B C 7 0 0 YPH1477 Y B C 5 3 4 Y B C 5 3 5 Y819 Y B C 5 6 4 Y B C 5 6 5 Y B C 5 8 3 Y B C 5 8 4 Y B C 5 8 5 Y B C 6 1 9 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 cep3-l Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 ctfl3-30 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 slkl9A::kanMX6 Mata 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 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 ctf3A::TRPl Mata rad61A::HIS3 cin8A::kanMX6 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 sgtl-5::LEU2 Mata ura.3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 skpl-3::LEU2 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 rad61A::HIS3 skpl-4::LEU2 Mata ade2-l trpl-1 canl-100 his3-ll,15 leu2::LEU2tetR-GFP ura3::3xURA3tet0112 PDS1-13MYC-TRP1 Mata ade2-l trpl-1 canl-100 his3-ll,15 leu2::LEU2tetR-GFP ura3::3xURA3tetOU2 PDS1 -13MYC-TRP1 rad61A::HIS5 Mata ade2-l trpl-1 canl-100 his3-ll,15 leu2::LEU2tetR-GFP ura3::3xURA3tet0112 PDS1-13MYC-TRP1 rad61A::HIS5 Mata trpl-1 :lacO-TRP-LEU2 his3-ll,15:lacR::GFP-HIS3 leu2-3,112 ura3-l ade2-l canl-100 Mata trpl-1 :lacO-TRP-LEU2 his3-ll,15:lacR::GFP-HIS3 leu2-3,112 ura3-l ade2-l canl-100 rad61A::kanMX6 Mata 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 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 Mayer et al., 2001 This study This study Mayer et al., 2001 This study This study K . Nasmyth K . Nasmyth K . Nasmyth K . Nasmyth 87 Table 3-1 List of yeast strains used in this chapter, Y B C 6 8 8 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 This study Y B C 6 8 9 Mata 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 This study Y B C 6 9 0 Mata 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 This study Y B C 6 9 1 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 rad61A::HIS3 containing Y C p l a c l W-dedl-51 This study Y B C 6 9 2 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 rad61A::HIS3 containing Y C p l a c l 1 \-dedl-54 This study Y B C 6 9 3 Mataura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 dedlA::kanMX6 rad61A::HIS3 containing Y C p l a c l 1 \-dedl-55 This study Y B C 6 9 4 Mata 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 This study Y B C 6 9 5 Mata 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 This study YH1444 Mata ade2 trpl leu2 canl his3-l l,15::GFP(pAFS144,thermo- Goshima and stable)-Lad-HIS3 leu2-3,112 ura3-l CEN 15(1.8 kb)-LacO- Yanagida., URA3 2000 Y B C 6 9 6 Mata ade2 trpl leu2 canl his3-l l,15::GFP(pAFS144,thermo-stable)-LacI-HIS3 Ieu2-3,U2 ura3-l CEN 15(1.8 kb)-LacO-URA3 dedlA::kanMX6 containing Y C p l a c l W-dedl-51 This study Y B C 6 9 7 Mata ade2 trpl leu2 canl his3-ll,15::GFP(pAFS144,thermo-stable)-Lad-HIS3 leu2-3,112 ura3-l CEN15(1.8 kb)-LacO-URA3 dedlA::kanMX6 containing Y C p l a c l W-dedl-51 This study Y B C 7 0 1 Mata ade2 trpl leu2 canl his3-ll,15::GFP(pAFS144,thermo-stable)-LacI-HIS3 leu2-3,112 ura3-l CEN15(1.8 kb)-LacO-URA3 rad61A::HIS3 This study 88 Table 1 List of yeast strains used in this chapter, continued Y B C 6 0 3 M a t a trpl ade2 ura3 leu2 RAD61 -13MYC-TRP1 SCC3- This study 3HA-HIS3 Y B C 6 0 4 Mata trpl ade2 ura3 leu2 RAD61-13MYC-TRP1 SCC3-3HA- This study HIS3 Y B C 6 0 7 Mata trpl ade2 ura3 leu2 RAD61-13MYC-TRP1 SMC1- This study 6HA-HIS3 Y B C 6 0 8 Mata trpl ade2 ura3 leu2 RAD61-13MYC-TRP1 SMC1- This study 6HA-HIS3 Y B C 6 1 1 Mata trpl ade2 ura3 leu2 RAD61 -13MYC-TRP1 SMC3- This study 6HA-HIS3 Y B C 6 1 2 M a t a trpl ade2 ura3 leu2 RAD61-13MYC-TRP1 SMC3- This study 6HA-HIS3 Y B C 6 4 9 Mata trpl ade2 ura3 RAD61 -13MYC-TRP1 SCC2-6HA-HIS3 pep4A::LEU2 This study Y B C 6 5 0 M a t a trpl ade2 ura3 RAD61-13MYC-TRP1 SCC2-6HA- This study HIS3 pep4A::LEU2 Y B C 5 8 9 Mata ura3 trpl ade2 leu2 rad61A::HIS3 SMC3-6HA-HIS3 This study Y B C 6 7 7 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 AL Tl - VFP-kanMX6 This study Y B C 6 7 8 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 DED1-13MYC-kanMX6 This study Y B C 6 7 9 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trpl A 63 DED 1-13MYC-ka nMX6 This study Y B C 6 8 0 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 This study VPS3-13MYC-kanMX6 Y B C 6 8 1 M a t a ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 VPS3-13MYC-kanMX6 This study Y B C 6 8 2 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 DEDl-13MYC-kanMX6 RAD61-VFP-kanMX6 This study Y B C 6 8 3 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 DEDl-13MYC-kanMX6 RAD61-VFP-kanMX6 This study Y B C 6 8 4 Mata ura3-52 lys2-801 ade2-101 his3A200 leu2Al trplA63 DEDl-VFP-kanMX6 This study Y B C 6 8 5 Mata 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 This study Y B C 6 8 6 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 This study Y B C 6 8 7 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 This study 89 R e s u l t s rad61A has genetic i n t e r a c t i o n s w i t h k i n e t o c h o r e a n d c o h e s i o n gene m u t a t i o n s 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) C S L rad61 A ndd 0-42 2 S L rad61 A kar3 A SL rad61 Achl4A (YBC329) C S L rad61 A ctf 19 A (YBC423) C S L rad61 A scc2-4 SL rad61 A smc3-42 SL 1 C S L - conditional synthetic lethality - the double mutant can survive at 25°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 non-essential gene deletion mutants are inviable in combination or a non-essential 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 19-central kinetochore CIN8, KAR3 - microtubule motors SCC2 - Cohesin loading protein SMC3 - subunit of core cohesion complex 90 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 in 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 of a double heterozygote diploid "pool" that is heterozygous for both the deletion mutant of 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 in Mat 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 in the pool of all possible double mutant combinations with the query strain w i l l be selectively lost from the population upon outgrowth. Each of 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 by P C R , labeled with Cy3 (rad61A) or Cy5 (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" of each of the deletion mutants in the two pools indicates the synthetic interaction. 91 •~ I HETEROZYGOUS DIPLOIDS MAI Hi: XXX/m.\ KtnMX TRANSFORM^ OAML • LEW? • HfAtm • HIS} • CANIR SELECT Oft SC LEU CONVERTIBLE HETEROZYGOUS otnoios .=? MATalu S X X X / mti MrrMX SS CANUcants LEU2-MFA1pr-HI$3 CONVERTIBLE HETEROZYGOUS OIPLOiD POOL g I HETEROZYGOUS OIPLOrOS POOL N W W « —. YtGUylgn URA3 SB xxx / m i kaoMX SS C A / V f / c a n U LEU2-MFAlpr-MS3 SPOftULATE , SELECT ON MAGIC MEDIUM IMM V«'»HAPLOIDS • — - M A T * = > / f j | l A U f i A J o r — Y F G f * • = U H A ktnUX = a « w f A - LEU2-UfA1pr-HIS3 CONTRCH EXPEKIMEN1AI MM »FOA MM -URA Single mutants DouMe mutants ANALYZE WITH MICROARRAY r i f t Red TAG »pot* indiane Y K O * synthetic lethal with query YKO Orange TAG spots suggest synthetic fitness defect From Pan etal., (2004) Figure 3-1 - Schematic of 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 of strains is analyzed with a microarray. 92 See Table 3-3 for the results o f the screen. We performed random spore analysis on a subset of the results and found in all instances that the synthetic interactions were re-confirmed (6/6 interactions tested by random spore analysis were re-confirmed, see Table 3 for the deletion mutants tested). We 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 in the establishment of 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 Scclp) as well as CHL1, a D N A helicase that is involved in 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) S G O l CHL4 RSC1 CSM3 CTF4 BRE2 M C M 1 6 CTF18 M G A 2 YPL017C YLR111W CTF8 DUN1 YDR018C RAD27 • * Tested and confirmed with Random Spore Analysis Genes involved in chromosome segregation Cohesion LST4 LSM1 DCC1 94 Query Strains ICTF8 CTF4 HRC1 P0L32 RAD 2 7 ELG1 DDC1 Deletion Mutant Array 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 in cohesion so we were interested in 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 in either G l phase (before D N A replication has occurred) using the mating pheromone alpha-factor, or in 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" in 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 wi ld type strains, the percentage o f cells with two distinguishable G F P signals in 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 ld type cells arrested with nocodazole represent a basal rate of breathing o f the paired chromatids. This assay was performed for two sites on chromosome arms as wel l 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 of 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, protein-D N A interactions are fixed using formaldehyde treatment (to cross-link protein-DNA interactions). The cells are then lysed and the cell lysate is sonicated in order to shear D N A to 300 to 500 base pairs in 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 in order to reverse the formaldehyde cross-linking; D N A is subsequently isolated by phenol extraction. Analysis of the D N A fragments that immunoprecipitated with the protein of interest can be assessed by P C R using primers flanking the region of interest. Although we could clearly see cohesion D N A in 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 by co-immunoprecipitation experiments performed on yeast extracts. We performed A n t i - H A IPs using yeast extracts from strains that carried either endogenously tagged SCC3-3HA, or SMC1-6HA, or SMC3-6HA individually or in combination with endogenously tagged RAD61-13MYC. We could not detect any association of Rad61p with 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 in loading of cohesin onto chromatin, we performed chromosome spreads in both wi ld type and rad61A strains that expressed an epitope tagged cohesin component (Smc3-6HAp). Cohesin is loaded onto chromatin in G l (after release from alpha-factor arrest) and dissociates in anaphase. rad61A strain background had no effect on the kinetics o f loading or the dissociation o f cohesin (Figure 97 3-3d). We examined -50 nuclei at each of the time points in Figure 3-3d and representative images of Smc3-6HAp on or off chromatin for wi ld 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 co-immunoprecipitation assay on yeast extracts prepared from two strains that had endogenously tagged Rad61-13MYCp and Scc2-6HAp. We found that there was no detectable association between the two proteins in 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. Many o f the genes have a defect in sister chromatid cohesion that is comparable to that observed in 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 in the establishment of cohesion. 98 a) o. 15 Ik a ~ 10 * 5 Sister chromatid cohesion assay i^HHIBIiBHHflilHBIi^H t Nocodaale j alpha-factor Chromosome V, 35 Kb from CEN i iI. i il,, Nxodasle | alpha-factor Chromosome IV, right arm alpha-factor Chromosome XV, 1.8 kB from CEN Anti-HA IP 1 2 3 4 5 6 7 8 9101112 c) Anti-HA IP Rad61- Scc2 13MYC 6HA T S I P T S I P T S IPT S IP Rad61-13MYC Scc2-6HA #1 #2 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 1 2 3 4 5 6 1 2 3 4 7 8 9 1011 12 5 6 7 8 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 SMC3-6HAp rad6m alpha-factor arrest alpha-factor arrest alpha-factor arrest re lease-30 min release - 75 min RAD61-13MYC Untagged SMC3-6HA TOT IP TOT IP TOT IP Scc2-6HAp Rad61-13MYCp Cohesin binding site - Chromosome V - 5 4 9 7 Kb Control site - POL1 - YNL102W 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 ) (YPH1477, YBC534,535) , Chromosome IV arm (Y819, YBC564,565), and Chromosome X V (1.8 kb from C E N ) (YPH1444, Y B C 7 0 1 ) . B) A n t i - H A Co-IP on yeast extract expressing Rad61-1 3 M Y C p and Scc3-3HAp (lanes 1 and 2) (YBC603) , Scc3-3HAp (lanes 3 and 4) (YBC583) , Rad61-13MYCp and S m c l - 6 H A p (lanes 5 and 6) (YBC607) , S m c l - 6 H A p (lanes 7 and 8) (YBC584) , Rad61-13MYCp and Smc3-6HAp (lanes 9 and 10) (YBC611) , or Smc3-6HAp (lanes 11 and 12) (YBC585) . 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 Rad61-13MYCp (upper panel lanes 1 to 3, bottom panel lanes 1 and 2) (YBC198) , Scc2-6HAp (upper panel lanes 4 to 6, bottom panel lanes 3 and 4) (YBC619) , and two strains expressing Rad61-13MYCp 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 , Smc3-6HA) and (YBC589 , Smc3-6HA rad61A). We performed chromosome spreads using A n t i - H A primary and a fluorescent secondary ( G A N - F ) at 1/1000 dilutions. Smc3-6HA appears red and D A P I appears green. A t each of the time points we examined ~50 nuclei, and representative images are shown. E) ChIP P C R assays on strains expressing Rad61-13MYCp (YBC198) , Smc3-6HAp (YBC585) , or untagged control strain (YPH499) using primers for a cohesin binding site and POL1 as a non-cohesin binding site control. By ChlP-chip analysis Rad61p associates with regions around centromere DNA In an effort to understand Rad61p's role in binding chromatin we sought to determine i f the chromatin binding occurred at specific loci on chromosomes. We performed a Chromatin Immunoprecipitation followed by microchip analysis (ChlP-chip experiment) in collaboration with Michael Snyder's laboratory at Yale 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 of 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 in 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 Rad61-13MYCp immunoprecipitated D N A sample over the untagged control immunoprecipitated D N A sample with a threshold that must be met in 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. We found that there was specific binding to a majority of the centromere regions (12 of the 16 chromosomes) in budding yeast with little other specific binding sites in the rest o f the genome (Figure 3-4a). Confirmation P C R s were performed using template D N A from Rad61-13MYCp 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 in Michael Snyder's laboratory at Yale 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 Rad61-13MYCp 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 in 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 in sonication treatment of the yeast lysate could shear D N A to different average sizes and this would affect the sequences that could be detected in the analysis. The nuclear localization of 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, Chrom IV Chrom VIII, IX, X Chrom XI, XII i . B, t 1 1 Chrom V, VI, VII " *• * ' i . a t k i i t i t i i i • CEN t > > ttt t 1 , i J t i l l t i t i t 1 t l. t i t Chrom XIII, XIV Chrom XV, XVI b) t t tt YBC198YPH499 YBC198YPH499 YBC198YPH499 RAD61- Uh- RAD61- Un-13MYC tagged 13MYC tagged C E N 1 6 CEN2 RAD61- Un-13MYC tagged 1 M # t t c) YBC198 YPH499 YIP412 RAD61-13MYC Untagged NDC10-13MYC Total IP Total IP Total IP T IP T IP T IP HMR CEN3 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 wi ld 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 Rad61-1 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 Rad61-1 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 in 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. We 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. Mark Flory in Rudi Aebersold's laboratory at the University o f Washington performed the tandem mass spectrometry analysis. We were consistently unable to identify more than 2 peptides o f Rad61p in the elution mixture. We decided to modify our approach and expressed a GST-Rad61 construct in E. coli, purified the construct with glutathione agarose beads and then incubated the beads with budding yeast lysate in order to find potential interactions. We were able to recover approximately 200 peptides o f Rad61p as wel l as other peptides specifically in the G S T -Rad61 experiments versus G S T alone ( A l t l p , Ded lp , Vps3p, Kar2p, Snul3p, Tfp lp , and Tublp) (see Table 3-4). We 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 of 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). We did not detect interactions between Rad61p and Vps3p (Figure 3-5a, lane 9 in the upper panels, and lane 18 in the bottom panel) or with Rad61p and A l t l p (Figure 3-5b, see lane 6). A peptide of D e d l p had also been identified previously in 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 in the initiation of 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 in 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 in budding yeast) and therefore it may have additional functions inside the nucleus (Honey et al., 2001). We 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 in budding yeast cells (Figure 3-5c, see lane 6). In fission yeast there is intriguing evidence that D e d l p could be post-translationally modified and that translation o f other proteins could be affected in response to different forms o f cellular stress (L iu et al., 2002). We decided to check the 104 interaction between Rad61p and D e d l p when cells have undergone D N A damage induced by M M S . We found that the interaction between Rad61p and D e d l p did not detectably change in cells that had been treated with M M S (data not shown). a) YBC678 YBC680 b) c) DED1-13MYC VPS3-13MYC GAL-HA- GAL GAL- GAL HA-RAD61 control HA-RAD61 control T 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 9 1011 12 DED1-13MYC Gal-HA-RAD61 Tot Sup IP 13 14 15 YPH499 GAL- GAL HA-RAD61 control VPS3-13MYC Gal-HA-RAD61 Tot Sup IP 16 17 18 YBC677 ALT1-VFP GAL- GAL HA-RAD61 control HA-RAD61 T IP T IP T IP T IP HA-RAD61 ALT1-VFP Tot Sup IP 7 8 YBC682 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 in 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 in lower panel), or V P S 3 - 1 3 M Y C with a G A L control plasmid (lanes 10 to 12 in upper panels), were grown in galactose (2%) to induce HA-Rad61p 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. Lower 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 in 2% galactose to induce HA-Rad61p expression. Yeast extracts were then prepared and A n t i - H A IPs were carried out. An t i -G F P and A n t i - H A Westerns were performed on the samples. C) Yeast extracts from strains expressing Rad61-VFP (lanes 1 to 3), or Rad61-VFP 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 in initiation of 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 of 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 in 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 H4 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 Ded lp by 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 C P Y 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 of 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 of D e d l p in each fraction. Acetylated H4 and C P Y were used as controls for chromatin and cytoplasmic proteins. B) Live cell fluorescence microscopy on D e d l - V F P cells. Cells were immobilized by low melting point agarose (see Materials and Methods). We examined -100 cells and this a representative image using one focal plane. 107 DED1 is an essential gene in budding yeast and certain temperature sensitive alleles have been shown to cause both translation and splicing defects at the non-permissive temperature. We were interested to see i f temperature sensitive alleles o f DED1 may have defects in 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-54, dedl-55, dedl-56, dedl-57, and dedl-58). We 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). We also transformed the plasmids into another diploid strain that had one copy of DED1 gene deleted and that was homozygous null for RAD6L After sporulation of 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 by the different dedl mutant alleles are shown in Table 3-5. A l l strains containing a combination of mutant alleles o f dedl and rad61A were able to grow. However, the restrictive temperatures of strains containing two temperature sensitive alleles, dedl-51 and dedl-57, in combination with rad61A, were lowered, indicating a genetic interaction between the mutations of 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 of the non-essential chromosome fragment) between the strains containing the dedl mutant alleles and a wi ld 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 of red sectors compared to the wi ld type strain (Table 3-5 and Figure 3-7). dedl-55 mutant strains contained 238 half-red sectors in a population o f 6018 colonies counted (3.95%) versus 18 half-red sectors in a population o f 4113 wi ld type colonies counted (0.44%) representing an increase o f approximately 9x that of wi ld 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 C-terminal 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 Figure 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 wi ld type. B y half-sector analysis, dedl-55 strains lose the chromosome fragment at a rate ~ 3 to 4x that o f w i ld 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 in order to assess precocious sister chromatid separation. Cells were arrested with a-factor and released into media containing nocodazole either at 30°C (permissive temperature) or 37°C (restrictive temperature). A t 15 minute time points cell cultures were shifted from 30°C to 37°C, in 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 in 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. We 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 of Ded lp to chromatin (Figure 3-6a) suggests that 110 this S phase progression delay could be a direct consequence o f inactivating Ded lp . Alternatively, the delay could be caused by aberrant transcription or translation o f G l or S cyclins similar to a temperature sensitive allele found in hamster B H K 2 1 cells, ts ET24 cells, with a mutation in D D X 3 X (a D E A D - b o x gene), which caused a G l / S arrest with a decrease in the m R N A of cyclin A (Fukumura et al., 2003). A mutant defective in the translation initiator eIF4E in budding yeast, which caused a G l to S phase progression defect, was also rescued by expression of Cln3p (Danaie et al., 1999). There were very few large budded cells with duplicated D N A in 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 in sister chromatid cohesion in dedl-51 strains at 37°C as approximately half of 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). I l l a) LOG Alpha-factor Nocodazole 30°C Nocodazole 37°C 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 Nocodazole 60 min 30°C Then to 37°C 1 1 j J i — L U L L J b) DIC GFP 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 wi ld type (YPH1444) and a dedl-51 (YBC696) ts mutant arrested in 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 G2 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 of 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 of 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 in our diploid-S 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 of the synthetic lethality profile o f rad61A led us to test for cohesion defects in rad61A strains because rad61A clustered next to mutants in cohesion genes. We indeed observed a cohesion defect but were unable to show any physical interaction of 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 in rad61A strains. Rad61p may be involved in 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 Rad61-13MYCp expressed at endogenous levels. Using the ChlP-chip assay, Rad61p bound specifically at regions clustered around centromeres. Extensive work purifying the components of the yeast kinetochore in recent years has not identified Rad61p as a component of the budding yeast kinetochore (De W u l f et al., 2003; Measday and Hieter, 2004). We 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 in the ChlP-chip assay is longer than that used in our follow-up experiments (Figures 3-4b and 3-4c) and this may have an effect in 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 of the cohesin complex association with centromere regions (Lengronne et al., 2004; Glynn 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 of Rad61-13MYCp. The eluted samples were purified and the proteins digested with trypsin, with the tryptic peptides subjected to tandem mass spectrometry analysis. We were consistently unable to purify more than 2 peptide fragments of 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. We did however isolate one peptide fragment of 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. While we were able to see intact Rad61-13MYCp by western blotting of 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 in E. coli, purifying the protein using glutathione agarose beads and then incubating the beads with yeast lysate. We 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 in the GST-Rad61 and not in the G S T alone control experiment. Testing the top three results confirmed that Rad61p did indeed bind to one of these proteins, Ded lp , 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. We had performed the initial characterization o f the binding of D e d l p and Rad61p using an over-expressed HA-Rad61 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 Asp-Glu-Ala -Asp ( D E A D ) box (Rocak and Linder, 2004). Along with the D E A H and DExH-box families they form a large super-family o f helicases that are highly conserved and are present in 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 of 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 in budding yeast and they have been implicated in 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 in an A T P dependent manner (Fairman et al., 2004). Ded lp ' s role in initiating translation is well established and a dedl cold sensitive allele shows defects in global protein synthesis when shifted to non-permissive temperature. A decline in polyribosome levels occurs almost immediately upon shift to lower temperatures consistent with a direct role of D e d l p in 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 ' -UTR) 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 in our strain background, and consistent with its role in 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). Using splicing-sensitive microarrays ( D N A chips that contain sequences that correspond to intronic sequences and to sequences at 5' and 3' UTRs) , 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 in 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 in chromosome segregation? It seems likely that any role would be direct, as both proteins bind chromatin and can physically interact. The genetic interactions between mutant alleles o f dedl and rad61A and also of rad61A and mutants in kinetochore and sister chromatid cohesion function, are consistent with a direct role in chromosome segregation. The biochemical function of the two proteins has not been elucidated and there remains the possibility that the role o f D e d l p in chromosome segregation is in 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 in those two processes could contribute to chromosome loss through indirect action on genes involved in 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 by deleting the intron of ANC1, a gene that is implicated in cell cycle control (although the exact function of 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 in typical cells (many ribosomal proteins contain an intron as wel l as genes coding for actin and tubulin) (Ares et al., 1999). Using microarray chips that contain intronic sequences (the splicing-sensitive chips mentioned above), researchers have recently been able to determine effects of deleting specific genes involved in splicing and which 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 in m R N A metabolism) can be assessed. Some of the genes that have introns that are interesting in terms of potential chromosome segregation defects and/or cell cycle control defects include GLC7, a phosphatase that de-phosphorylates Ip l lp substrates, MOB1, part o f the mitotic exit network, TUB1 and TUB3, coding for alpha-tubulin, ACT1 coding for actin, and PH085 involved in cell cycle control (Saccharomyces Genome Database, SGD) . Ded lp ' s role in translation initiation could be another potential way that D e d l p has an indirect role on chromosome segregation. Proteins that are involved in cell cycle regulation, kinetochore function and cohesion could be potentially reduced in a dedl hypomorph mutant. In the fission yeast S. pombe, the homologue of D e d l p has been found to associate with Cdc2 (the C D K in fission yeast) and with C h k l (a protein kinase that is involved in response to D N A damage) (L iu et al., 2002). It was shown that D e d l p could be post-translationally modified in 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 in response to cellular stress (L iu et al., 2002). A n attractive idea would be that Ded lp is affecting translation o f proteins that are needed to respond to the cellular stress of damage. In budding yeast, a mutant allele o f DED1, dedl-18, has been found to impair brome mosaic virus ( B M V ) R N A 2 translation while still maintaining general translation of other m R N A and allowing growth (Noueiry et al., 2000). D e d l p could either have a direct effect in 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 in the B M V R N A 2 m R N A to allow translation initiation (Noueiry et al., 2000). 118 Could 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 in initiating translation, although it may have a role in translating a subset o f proteins that are important for chromosome segregation. Testing the expression levels of 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 of cohesin complex members and some members o f the kinetochore complexes and have found no detectable difference in expression levels between wi ld 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 in rad61A cells versus wi ld 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 of Ded lp 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 Ded lp 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 of the proteins are interacting. Ded lp ' 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 of Rad61p in the cytoplasm that cannot be detected by microscopy. The conditional synthetic lethality between rad61A and temperature sensitive alleles of dedl points to the importance o f the interaction between the two proteins. Preliminary data showing sister chromatid cohesion defect in 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 in chromosome segregation, and in particular, kinetochore function, we used SGT1 as a genetic entry point. Sgt lp is a protein partner o f Skp lp and plays a role with Skp lp at the budding yeast kinetochore and the S C F complex. We were interested in finding proteins that could play a role in regulation o f the kinetochore and since Sgtlp plays a role in activating the C B F 3 complex, we reasoned that it would be a suitable entry point. We attempted two large scale screens, a high copy suppression screen and a genome wide two-hybrid screen, and we identified a novel factor in chromosome segregation, Rad61p, that is apparently not directly involved in Sgtlp function, but is important in 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 in chromosome segregation was a genome wide two-hybrid screen using SGT1 as the "bait". We were able to identify genes encoding known protein partners o f Sgt lp, including Skp lp 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 Sgt lp function. We were especially interested in Rad61p, because the deletion mutant o f the gene displayed a chromosome missegregation phenotype. We were unable to detect an interaction between Sgtlp and Rad61p by 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. We found 122 when we performed a ChlP-chip analysis with long fixation times using formaldehyde to cross-link DNA-protein interactions that Rad61p could bind preferentially to regions close to centromeres. The two-hybrid interaction may represent an indirect interaction between Rad61p and Sgtlp 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. When we investigated the morphology of the cells, we found that rad61A strains contained more cells with short spindles and the nucleus at the neck, compared to wi ld type cells. This was indicative o f a delay in progressing through the metaphase to anaphase transition consistent with the delay depending on Mad2p. Mutants displaying a similar phenotype have been found by mutation of genes encoding proteins involved in processes such as kinetochore, microtubule, and sister chromatid cohesion function (Hyland et al., 1999; Mayer 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 in 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). We explored the possibility that Rad61p could be involved in 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 by Rad52p foci formation. 123 Rad61p and its role in sister chromatid cohesion Using 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 by 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 of non-essential proteins that when deleted have a moderate sister chromatid cohesion defect. Many o f these proteins have been provisionally categorized as proteins involved in establishing sister chromatid cohesion during or immediately after D N A replication because they are not part of the core cohesin complex, are not needed to maintain cohesion once established, and are not involved in loading cohesin onto chromatin. The D N A damage sensitivity o f radSIA mutant strains may be the result o f the moderate defect in 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). We 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; Unal et a l , 2004). Cohesin is recruited to the sites of double strand break and this recruited cohesin is functional for sister chromatid cohesion (Strom et al., 2004). How sister chromatid cohesion is generated outside o f S phase remains unclear. Establishment of cohesion may simply depend on proximity of the sister chromatids provided in S phase 124 immediately behind the replication fork and in G2 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 of the non-essential genes involved in 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 of any of the non-essential complexes that are important for sister chromatid cohesion (Hanna et al., 2001; Mayer et al., 2001; Mayer et al., 2004; Warren et al., 2004). Identifying proteins that interact with Rad61p could shed light onto its role in sister chromatid cohesion. Rad61p and Dedlp interaction We incubated a large amount of 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. We tested the top three "hits" as determined by the number of 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 of 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 in R N A metabolic processes such as splicing, ribosome bio-125 synthesis, and translation initiation. We showed that a portion o f D e d l p binds to chromatin in addition to the documented localization in 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 of the temperature sensitive alleles o f DED1 (dedl-55) exhibits an increase in chromosome missegregation at a semi-permissive temperature, as evidenced by a nine fold increase in 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 of 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 (Scclp 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 of 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 wel 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 in addition to its roles in translation and splicing. There are examples of 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). Ded lp could be playing multiple roles in the budding yeast cell in 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 of 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 in the Homo Sapiens non-redundant peptide sequence database (Abdelhaleem et al., 2003). The functions of 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 in 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). Many o f the D E A D - b o x family members remain un-characterized but two important characteristics of the D E A D - b o x family members that have been investigated have been described; there is a dysregulation o f expression in cancer and an involvement in 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 wel l as other cancers and down-regulated in 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 in 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; Yang et al., 2003b). DDX10 was found to be involved in a leukemia-associated chromosomal translocation Inv l I(pl5q22) generating an in-frame fusion of 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 in humans ( D D X 3 X , D D X 3 Y , and D D X 4 1 ) do not have documented roles in cancer and tumourigenesis, Ded lp is also highly homologous to the other D E A D - b o x family members (especially in the nine conserved domains). The accepted role of human R N A helicases in 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 of oncogenes and/or tumour suppressor genes could lead to tumourigenesis and altering R N A helicase activity (either by mutations, deletions of the gene, or by overexpression) could be one o f the "hits" needed for tumour progression. We have shown evidence that a temperature sensitive mutation in budding yeast DED1 can cause an increase in the rate of 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 in chromosome segregation. Finding the gene that 128 -I is responsible for the loss of the chromosome fragment based on its altered expression in 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 in colorectal cancer cell lines that are both aneuploid and overexpressing R N A helicases. Could R N A helicases be playing a more direct role in 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 Dedlp) 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 (Kikuma 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 , in fission yeast extracts (Liu et al., 2002) and that DED1 has genetic interactions with CDC2 ( C D K in fission yeast). In budding yeast D e d l p has been identified as a protein interactor with Cdc28p. We also identified Ded lp as a protein interacting with a nuclear localized protein, Rad61p. A fraction of both Rad61p and D e d l p binds chromatin. These results suggest a potential role for Ded lp on chromatin but definitive proof of 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. LOC57821 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). LOC57821 has been identified as one o f the putative homologues o f ctf genes (specifically o f RAD61) that are candidates for sequencing in 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 of 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 in cooperation with P K B . Conclusion Rad61p was initially identified as a potential protein partner o f Sgtlp based on a genome wide two-hybrid screen using SGT1 as the "bait". Rad61p and Sgt lp did not detectably interact in 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 of endogenous chromosomes to ~100x that of wi ld type strains. The chromosome 130 Figure 1 - Putative Human homologues of Rad61p in Human Cells a) RAD61 31 IfDlSsViPJgVSlTlssllAD^ LOC57821 105 fflES^ElQKfflLEEEEDEEWRSM|Kl|C^KLfflQNQEPVND^EBKLKF^QL|DL RAD61 89 @NV@|l|SSSg[rS MA^KlJEKJgSgFNFgDGSKASKR^RgTYQK^ANgTS LOC57821 162 E I P B - - -LgjTfflsaY^NEgNgGKlSQLC@SNDF|Q|i^fflLS|TNGSCEENK-RAD61 139 SiE@DVQD^^l3M^E^SlSKSND|NEFI§KLPR |D^^NKfLENEMKMDDS LOC57821 214 --DRpHLVE^GEELLNLQD^SlGILPPlfcANSlSNfflpQQLLPRSSfflSSVfaGT RAD61 195 IENNggRTSggKggGKFRTILgNK^ KEfJEIMGE§\/gQKANTLSLN§ADNgNA LOC57821 268 K ^ D s B A K l f f l A V T H S S T^PfAH 290 RAD61 247 E^GLgSTNBJYNELKNM^TgKg 269 b) RAD61 60 Hpf^AQDlsS^DGANEKPsBQ--LD|KRN|QBKilTSSgS -M0FfWDEIOjSAFfSFLD KIAA1212 671 BN^BLKKflaLD^BKMLTFOLF^LEKENfioLDwE^tfElSRRNV^^LKcSRr^MAoBnLEBKETj RAD61 117 GHKASKR^RRTYQ^DANinss|raPDVffl--raElsIT |HNEFrasiRraiYSD|NEF|L KIAA1212 731 EgEKEQJ^GLELL^SFKKgERg^SY^LSlaNQRB^ RAD61 172 KgPRADDD|LNK|l^NE|KMDDSIENNSIRTSl^]KKYGKFRTILl|KNf^gElSGE KIAA1212 78 8 DgEMENQTgQKMjE@LKflSSKRLEQLEKENK§I^QETSQLEKDKK§LE^^KRgRQ RAD61 228 E VjEofflANfflSLfflADNsSAf^^GLfl 252 KIAA1212 844 QAIJIjgDT^ E E H v K I G p j^^NKg 868 Figure 1 - Putative Human homologues of 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 the N-terminal of each p r o t e i n . B ) Rad61p and KIAA1212 (APE) are 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 region, near the N-terminal of Rad61p and i n the middle of APE. 131 missegregation phenotype o f rad61A strains is the result of a defect in sister chromatid cohesion and Rad61p may play a role in 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, Ded lp , and this interaction is functionally significant as evidenced by genetic interactions between temperature sensitive alleles o f DED1 and rad61A. D e d l p is a multifunctional protein with characterized roles in initiating translation and also as a member o f the spliceosome. 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