REGULATION OF SPINDLE BEHAVIOR BY DNA REPLICATION AND DAMAGE CHECKPOINTS IN BUDDING YEAST SAURABH RAJENDRA NIRANTAR INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 REGULATION OF SPINDLE BEHAVIOR BY DNA REPLICATION AND DAMAGE CHECKPOINTS IN BUDDING YEAST SAURABH RAJENDRA NIRANTAR (B.Tech.(Hons), Indian Institute of Technology, Kharagpur) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I would like to express my deepest gratitude to Prof. Uttam Surana for his guidance and mentorship which helped me throughout my course of study. I am grateful to my PhD Supervisory Committee members Prof. Wang Yue and Prof. Mohan Balasubramanian for their advice and encouragement. I am thankful to my collaborators Dr. Vaidehi Krishnan and Dr. Zhang Tao, from whom I have learnt a great deal, for stimulating my interest and captivating discussions. Dr. Hong Hwa is owed my gratitude for her help and advice, especially in the beginning of my studies. Dr. San Ling was always helpful and generous, and provided invaluable advice on the preparation of this manuscript. Dr. Indrajit Sinha taught me to conduct 2D gel electrophoresis, for which I am very grateful. My colleagues in US Lab, Khong Jenn Hui, Karen Crasta, Yio Wee Kheng, Liang Hong Qing, Zhang Tian Yi, Joan Cher and past members of the lab made my stay very pleasant and extended a great deal of assistance. Finally I would like to express my gratitude to my wife Renuka as well as my parents for their constant support and understanding, without which this thesis could not have been completed. i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii SUMMARY vi LIST OF TABLES . ix LIST OF FIGURES . x LIST OF SYMBOLS . xii CHAPTER Introduction 1.1 Cell Cycle in S.cerevisiae . 1.1.1 Mitosis . 12 1.1.1.1 Metaphase to Anaphase Transition . 12 1.1.1.2 Mitotic Exit . 16 1.1.1.2.1 Fear Pathway . 19 1.1.1.2.2 MEN Pathway . 20 1.2 DNA Damage and Replication Checkpoints 21 1.2.1 Replication and Intra S Phase Checkpoints 23 1.2.1.1 Activation of Effector Kinase . 25 1.2.1.2 Downstream Functions of Activated Checkpoint . 34 1.2.1.2.1Stabilization of Stalled Replication Forks . 37 1.2.1.2.2 Inhibition of Late Origin Firing 40 1.2.1.2.3 Suppression of Recombination and Repair of Collapsed Forks . 41 1.2.1.2.4 Inhibition of Cell Cycle Progression 42 ii 1.2.1.2.4.1 Direct Regulation of Spindle Elongation by Checkpoint . 44 1.2.2 DNA Damage Checkpoint 46 1.2.2.1 DNA Damage Checkpoint Activation 46 1.2.2.2 Downstream Functions of the DNA Damage Checkpoint 49 1.2.2.2.1 Prevention of Anaphase . 49 1.2.2.2.2 Prevention of Mitotic Exit . 50 1.3 Focus of this Project 55 CHAPTER Materials and Methods 2.1 Materials . 56 2.2 Methods 66 2.2.1 Bacterial Strains and Culture Conditions . 66 2.2.2 Yeast Strains and Culture Conditions . 66 2.2.3 Synchronization of Yeast Cells . 67 2.2.3.1 G1 Phase Synchronization . 67 2.2.3.2 Early S Phase Synchronization 68 2.2.3.3 G2M Phase Synchronization 68 2.2.3.4 Telophase Synchronization 68 2.2.4 Genotype Manipulation and Verification Techniques . 69 2.2.4.1 Transformation 69 2.2.4.2 Genomic DNA Extraction . 69 2.2.4.3 Southern Blotting for Verification of Transformants 71 2.2.4.4 Diagnostic PCR . 71 2.2.5 Phenotype Analysis Techniques 72 2.2.5.1 Immunofluorescence . 72 2.2.5.2 Fluorescent Protein Procedures . 73 2.2.5.3 Microscopy 74 2.2.5.4 Fluorescence Activated Cell Sorting . 74 2.2.5.5 Protein Analysis 74 2.2.5.5.1 Extraction of Protein by Trichloroacetic Acid 74 iii 2.2.5.5.2 Extraction by Mechanical Lysis using Glass Beads . 75 2.2.5.5.3 Immunoprecipitation of Protein 76 2.2.5.5.4 Western Blotting . 76 2.2.5.5.5 Two-Dimensional Gel Electrophoresis . 77 2.2.5.5.6 Pulse Chase Assay 78 2.2.6 Recombinant Protein Expression and Purification 79 CHAPTER Direct Regulation of Spindle by DNA Replication Checkpoint 3.1 Introduction . 81 3.2 Checkpoint Mutants Elongate Spindle and Divide Nucleus in the Absence of Representative Mitotic Events 84 3.3 Mitotic Entry is Dispensable for Premature Spindle Elongation and Nuclear Division in mec1-1 . 89 3.4 Upregulation of Microtubule Associated Proteins Cin8 and Stu2 in Checkpoint Deficient Cells 94 3.5 Ectopic Expression of Cin8 Causes mec1-1 like Phenotype in Wild Type Cells . 98 3.6 Downregulation of Cin8 and Stu2 in mec1-1 Restrains Spindle Elongation 102 3.7 Role of Elongation-opposing Factors in Restricting Spindle Extension During Checkpoint Arrest . 106 3.8 Ectopic Expression of Effector Kinase Rad53 Causes Spindle Collapse 110 3.9 Discussion 114 CHAPTER Regulation of Spindle Dynamics by DNA Damage Checkpoint 4.1 Introduction . 120 4.2 Artificial removal of Cohesin does not Lead to Complete Segregation of Damaged Chromosomes in DNA-damaged Cells 122 4.3 Negative Regulation of Microtubule Associated Proteins Cin8 and Kip1 by DNA Damage Checkpoint . 130 iv 4.4 Role of Cdh1 in Regulation of Spindle Extension 134 4.5 Hypo-phosphorylation of Cdh1 in cells with Activated DNA Damage Checkpoint 138 4.6 Effect of Ectopic Cdc5 Expression on Spindle Dynamics in cdc13-1 Cells . 142 4.7 Mutation of Cdc5 Phosphorylation Sites on Cdh1 Prevents Spindle Elongation in Checkpoint Deficient Cells . 145 4.8 Discussion 149 Chapter Mechanism of Spindle Regulation by Replication Checkpoint 5.1 Introduction . 152 5.2 Precocious Spindle Elongation in mec1-1 cells can be Prevented by Inhibition of CDC28 (Cdk1) activity 153 5.3 Levels of Microtubule-Associated Proteins are lower in mec1-1 cdc28as1 Cells 157 5.4 Ectopic Inhibition of Cdc28 Destabilizes Cin8 and Kip1 . 161 5.5 Cdh1 is Responsible for Cin8 and Kip1 Destabilization upon Activation of Replication Checkpoint . 164 5.6 Interaction of Cdc28 and Cdc5 Kinases with Cdh1 . 168 5.7 Role of Cdc5 in the Replication Checkpoint . 172 5.8 Other Checkpoint Mediated Mechanisms for Restraining Premature Spindle Elongation 176 5.9 Discussion 180 Chapter Conclusions and Future Work 6.1 Regulation of Spindle Dynamics by DNA Replication Checkpoint . 183 6.2 Regulation of Spindle Dynamics by the DNA Damage Checkpoint 186 6.3 A Unified View of DNA Replication and Damage Checkpoints 187 Bibliography Appendix I Appendix II v SUMMARY High fidelity transmission of the genome to the next generation is crucial for the continued survival of all species. At the cellular level, this is accomplished by the sequential duplication and symmetrical segregation of the genome to two daughter cells, during the cell division cycle. However, the genetic information is vulnerable to multiple environmental factors such as free radicals and high energy radiation, which can result in the alteration of its information content with potentially catastrophic consequences. To counteract this possibility, cells have evolved surveillance pathways known as checkpoints to monitor genomic integrity. These pathways halt cell cycle progression upon detection of genomic insults and undertake ameliorative steps to repair detected damage. In the budding yeast Saccharomyces cerevisiae, as in human cells, checkpoints are active during all phases of the cell cycle monitoring various events. Replication checkpoint and DNA damage checkpoints are the two major surveillance pathways that monitor genome integrity. Replication checkpoint is activated in response to various forms of replication stress that impede the progression of the replication forks. The activated checkpoint helps to maintain the integrity of the stalled forks and causes cells to arrest with a short spindle and an undivided nucleus, thus preventing precocious segregation of chromosomes. This is consistent with the observation that cells defective in replication checkpoint, when treated with replication-inhibitors, arrest in early S phase but proceed to elongate their spindle and prematurely segregate the largely unreplicated vi chromosomes. It is generally believed that the checkpoint prevents premature segregation of unreplicated chromosomes by inhibiting precocious onset of mitosis. We began this work by testing this assumption. We find that chromosome segregation in checkpoint mutants is not accompanied by the characteristic mitotic events such as Cohesin cleavage or the activation of APC (Anaphase Promoting Complex). Our results strongly suggest that the replication checkpoint directly regulates spindle dynamics to restrain premature segregation of chromosomes. Hence, the untimely spindle elongation seen in checkpoint deficient mutants is not a consequence of premature entry into mitosis but a consequence of loss of this regulation. Given the substantial overlap between the effectors involved in replication checkpoint and DNA damage checkpoint , we enquired whether DNA damage checkpoint pathway also target spindle to restrain the segregation of damaged chromosomes until they are repaired. Our results suggest that DNA damage checkpoint uses two-pronged control to restrain chromosome segregation: (i) by inhibiting Cohesin cleavage and (ii) by preventing spindle elongation. Furthermore, we have uncovered the likely mechanism by which the DNA damage checkpoint regulates spindle behavior. 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Mol Cell. 2004 Dec 3;16(5):687-700. 2) Crasta K, Lim HH, Zhang T, Nirantar S, Surana U. Consorting kinases, end of destruction and birth of a spindle. Cell Cycle. 2008 Oct;7(19):2960-6. Epub 2008 Oct 13. 3) Tao Zhang¶, Saurabh Nirantar¶, Lim Hong Hwa, Indrajit Sinha and Uttam Surana. DNA damage checkpoint maintains Cdh1 in quasi-active state to prevent spindle elongation and Anaphase B. Dev. Cell. 2009 Oct; 17(4):541-51. ¶ : Equal Contribution. APPENDIX II Figure Wild-type and mec1-1 cells with epitope tagged Cin8 at native chromosomal loci (US4122 and US4123 respectively) were released from G1 arrest at 24oC into YEPD medium containing HU (20 mg/ml).Wild-type and mec1-1 strains with epitope tagged Stu2 at its native locus were treated similarly (US4128 and US4129 respectively). Samples were collected at 180 minutes and Cin8/Stu2 levels were estimated by Western blotting. Figure Wild-type cells with epitope tagged Cin8 and Stu2 (US 4122 and 4128 respectively) and wild-type with copies of GAL-RAD53-HA2 with epitope tagged Cin8 and Stu2 (US 4132 and 4131 respectively) were arrested in G1 phase using α-factor in YEP + Raff medium at 24oC for 150 minutes, followed by a further 60 in YEP + Raff + Gal while still arrested using α-factor. The cells were then released into YEP + Raff + Gal medium at 24oC, and samples were collected at 180 minutes for Western blotting. Figure GAL-PDS1 CIN8-HA3 (US4837), cdc13-1 CIN8-HA3 (US4687), GAL-PDS1 KIP1HA3 (US4824) and cdc13-1 KIP1-HA3 (US4678) cells were arrested in G1 phase using α-factor and then released into YEP + Raff + Gal medium at 32oC. Samples were collected at 90 minutes and processed for Western blotting. Figure mec1-1 and mec1-1 cdc28as1 strains expressing HA3 epitope-tagged versions of Cin8 and Kip1 (US4123, US4676, US6141 and US5896, respectively) as well as rad53-21 and rad53-21 cdc28as1 cells expressing HA3 tagged Cin8 and Kip1 (US4124, US4675, US6173 and US6174 respectively) were synchronized in G1 phase using αfactor and subsequently released into YEPD medium containing HU (20mg/ml) and 1NM-PP1 (500nM). Samples were collected at 120 minutes for Western blotting. Figure mec1-1 CIN8-HA3 (US4123) and mec1-1 cdc28as1 CIN8-HA3 (US5896) were synchronized in G1 phase using α-factor and subsequently released into YEPD medium containing HU (20mg/ml) but not 1NM-PP1. Protein levels as detected by Western blotting are shown. [...]... anaphase B in cells arrested in G2M due to activation of DNA damage checkpoint 129 x Figure 17 Checkpoint activation destabilizes plus-end kinesins Cin8 and Kip1 133 Figure 18 Spindle extension can be induced by Cin8 stabilization or Cdh1 deletion in DNA damaged cells 137 Figure 19 Phosphorylation status of Cdh1 and Cdc5 upon DNA damage checkpoint activation 141 Figure 20 Overexpression of Cdc5... in mec1-1 cells by downregulation of Cin8 and Stu2 105 Figure 13 Role of anti -spindle elongation factors Kip3 and Mad2 in replication checkpoint 109 Figure 14 Ectopic expression of effector kinase Rad53 causes spindle collapse even without checkpoint activation 113 Figure 15 Forced Cohesin cleavage in DNA- damaged cells fails to trigger anaphase B 126 Figure 16 Cohesin inactivation does... Precocious Spindle Elongation in mec1-1 cells is not strictly dependent on Clb1 and Clb2-Cdc28 Activity 93 Figure 10 Status of microtubule associated proteins Cin8 and Stu2 in checkpoint-deficient and proficient strains 97 Figure 11 Ectopic overexpression of Cin8 causes spindle elongation in wild-type cells arrested in early-S phase but not G2M 101 Figure 12 Prevention of spindle extension in mec1-1... de-phosphorylation of Tyr19 by the conserved tyrosine-phosphatase Mih1 (ortholog of human Cdc25) activates Cdc28 and enables Cdc28-Clb1/Clb2 complex to initiate mitosis (Booher et al., 1993) Another dimension to the regulation of Cdk-Cyclin complex is added by a class of proteins known as Cdk inhibitors In budding yeast, one of the prominent Cdk1 inhibitors is Sic1 which inhibits the activities of both S phase kinase... premature spindle elongation in DNA damaged cells 144 Figure 21 Effect of Cdc5-resistant Cdh1 expression on precocious spindle elongation in checkpoint deficient cells 148 Figure 22 Inhibition of Cdc28 kinase prevents premature spindle elongation in mec1-1 cells 156 Figure 23 Microtubule associated protein levels are diminished in mec1-1 cdc28as1 cells 159 Figure 24 Destabilization of Cin8 and. .. eventually cause the onset of S phase Similarly, Cdc28-Clb5/Clb6 is involved in the initiation of DNA replication and progression through S phase, whereas Cdc28Clb1/Clb2/Clb3/Clb4 facilitates progression through mitosis (Mendenhall and Hodge, 1998) (Figure 1) In budding and fission yeasts, the cell cycle is driven by a single Cdk i.e Cdc28/Cdc2 (Cdk1) in combination of various Cyclins In higher eukaryotes,... Kip1 checkpoint deficient cells with low Cdc28 kinase activity 163 Figure 25 Cdh1 is responsible for destabilization of Cin8 and Kip1 in response to replication checkpoint activation 167 Figure 26 Functional interaction of Cdc28 kinase and Cdc5 kinase with Cdh1 171 Figure 27 Role of Cdc5 in replication checkpoint 175 Figure 28 Delayed addition of HU prevents spindle elongation in mec1-1... the assembly of pre -Replication Complexes (pre-RCs), which recognize and bind to replication origins only during stages of low Cyclin-Cdc28 activity (i.e during G1), and permitting the initiation of replication only during high Cyclin-Cdc28 activity (Nguyen et al., 2001) During the late M-early G1 phases, hexameric complexes known as Origin Recognition Complexes (ORCs) composed of Orc1-6, bind to consensus... accumulating Microtubule-Associated Proteins (MAPs) Cin8, Kip1 and Ase1 breaks the bridge, allowing the separation of SPBs and assembly of a short spindle (Hoyt et al., 1992; Crasta et al., 2006) During G1 and early S phase, the abundance of Cin8, Kip1 and Ase1 is kept low by the ubiquitin ligase APCCdh1 which targets them for proteolytic degradation (Hildebrandt and Hoyt, 2001) An increase in the activity... Spo12 and Bns1 inhibit Fob1 to cause Cdc14 release The Cdc14 released by the FEAR pathway promotes the stability of the elongating anaphase spindle Pre-anaphase spindles are characterized by high microtubule turnover, while initiation of anaphase leads to a sudden Cdc14 dependent increase in spindle stability (Higuchi and Uhlmann, 2005) It is thought that Cdc14 promotes spindle stability by mediating . REGULATION OF SPINDLE BEHAVIOR BY DNA REPLICATION AND DAMAGE CHECKPOINTS IN BUDDING YEAST SAURABH RAJENDRA NIRANTAR INSTITUTE OF MOLECULAR AND. OF SINGAPORE 2009 REGULATION OF SPINDLE BEHAVIOR BY DNA REPLICATION AND DAMAGE CHECKPOINTS IN BUDDING YEAST SAURABH RAJENDRA NIRANTAR (B.Tech.(Hons), Indian. Negative Regulation of Microtubule Associated Proteins Cin8 and Kip1 by DNA Damage Checkpoint 130 iv 4.4 Role of Cdh1 in Regulation of Spindle Extension 134 4.5 Hypo-phosphorylation of Cdh1 in