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Orchestration of the Dna Damage Checkpoint Response Through the Regulation of the Protein Kinase Rad53

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Orchestration of the Dna Damage Checkpoint Response Through the Regulation of the Protein Kinase Rad53 ORCHESTRATION OF THE DNA DAMAGE CHECKPOINT RESPONSE THROUGH THE REGULATION OF THE PROTEIN KINASE RAD53 By Frédéric D. Sweeney A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular Genetics, University of Toronto © Copyright by Frédéric D. Sweeney 2009 ORCHESTRATION OF THE DNA DAMAGE CHECKPOINT RESPONSE THROUGH THE REGULATION OF THE PROTEIN KINASE RAD53 Frédéric D. Sweeney, Doctor of Philosophy, Graduate Department of Molecular Genetics, University of Toronto, 2009 ABSTRACT In order to maintain genome stability, DNA damage needs to be detected and repaired in a timely fashion. To cope with damaged DNA, cells have evolved mechanisms termed "checkpoints", where, upon damage, cells initiate a signal transduction cascade that results in the slowing or halting of the cell cycle, allowing efficient DNA repair. Defects in the DNA damage checkpoint result in an overall increase in genomic instability and are thought to fuel cancer progression. To facilitate our understanding of how DNA damage leads to cancer progression, it is crucial to fully comprehend how these signal transduction mechanisms function. In this work, we have characterized in great detail the mechanisms of regulation of Rad53 (a central regulator of the DNA damage response in Saccharomyces cerevisiae) at the genetic, biochemical and structural level. Firstly, we describe a complex biochemical two-step mode of activation of Rad53 by protein-protein interaction and multi-step phosphorylation. We also shed light onto the mechanisms by which Rad53 is turned off to allow the cell cycle to resume, a process termed DNA damage recovery and adaptation. We found that during adaptation, the polo-like kinase Cdc5 is required to attenuate Rad53 catalytic activity. Finally, the study of Rad53 at the molecular and atomic level revealed that in addition to being regulated through a complex network of protein-protein interactions, Rad53 autophosphorylation is orchestrated by a mechanism of dimerization, activation segment phosphorylation via A- loop exchange, as well as through an autoinhibition mechanism regulated by a specific α- helical region at the C-terminal extremity of its kinase domain. Such work is important in understanding the function of different proteins in DNA damage signaling. This knowledge will enhance our understanding of the progression of DNA damage related diseases such as cancer, and could eventually help in the long term the development of novel therapeutics as treatments against these conditions. ii ACKNOWLEDGEMENTS It is with tremendous humility that I present this work. During my graduate career, I realized early on that the greatest accomplishments are only possible with the help of highly motivated, intelligent, generous and resourceful people. I wish to thank the following people who have made this work possible: First and most importantly, my supervisor and great friend, Dr. Daniel Durocher who provided me with unprecedented support, giving me not only all the resources possible to perform my work, but also the moral support allowing me to take risks and to benefit from the rewards. I would also like to thank my committee members, Dr. Frank Sicheri and Dr. Grant W. Brown who guided me throughout these exciting times. Moreover, this work would have not been possible without the help, advice and support of my fellow lab members, particularly Sarah Jane Galicia, Dr. Pamela Kanellis and Dr. Rachel Szilard. Within all these individuals, I did not only find labmates but also mentors and friends. I also extend my appreciation to my collaborators, especially Dr. Nadine Kolas, Dr. Anne-Claude Gingras, Dr. Cynthia Ho, Genevieve Vidanes and Dr. David Tocyzski whose work was instrumental in many of my discoveries. Lastly, I would like to thank my parents, Pierre Sweeney and Johanne Demers Sweeney for their unconditional love and support. You have seen me grown intellectually and emotionally during this process and sacrificed a lot to provide me with all the tools necessary to succeed. Without you, this work would have never been possible and I strongly believe that we have earned this degree together. iii TABLE OF CONTENTS ABSTRACT...................................................................................................................................................ii ACKNOWLEDGEMENTS.............................................................................................................................iii TABLE OF CONTENTS................................................................................................................................iv LIST OF TABLES AND FIGURES...............................................................................................................vii LIST OF APPENDICES ..............................................................................................................................viii LIST OF ABBREVIATIONS .........................................................................................................................ix CHAPTER 1: DNA DAMAGE, GENOMIC INSTABILITY AND CANCER 1.0. DNA DAMAGE AND CARCINOGENESIS............................................................................................3 1.0.0 DNA damage in the lifespan of a cell..........................................................................3 1.0.1 How genomic instability leads to cancer progression..................................................4 1.1. SACCHAROMYCES CEREVISIAE AS A MODEL TO STUDY DNA DAMAGE RESPONSE.............................8 1.1.0 Saccharomyces cerevisiae as a genetic tool.................................................................8 1.1.1 Historical considerations..............................................................................................9 1.1.1.0 The discovery of the cell cycle genes 1.1.1.1 The discovery of the DNA damage checkpoint genes 1.1.2 S. cerevisiae DNA damage checkpoint genes and their mammalian homologs.........12 1.2. MECHANISMS OF DNA DAMAGE REPAIR AND CHECKPOINT...........................................................14 1.2.0 DNA Repair...................................................................................................................14 1.2.0.0 Recombination repair (HR and SSA 1.2.0.1 Non-homologous end joining (NHEJ) 1.2.0.2 Base excision repair and Nucleotide excision repair 1.2.1 Turning ON the DNA damage checkpoint response..................................................18 1.2.1.0 DNA damage checkpoints, an overview 1.2.1.1 The G2/M Checkpoint 1.2.1.2 The S phase Checkpoint 1.2.1.3 The G1/S Checkpoint 1.2.2 Turning OFF the DNA damage checkpoint response.................................................29 1.2.2.0 DNA damage checkpoint recovery 1.2.2.1 DNA damage checkpoint adaptation 1.2.3 The Rad53 protein kinase............................................................................................34 1.3. MODULAR PROTEIN INTERACTION DOMAINS...................................................................................37 1.3.0 The FHA Domain...........................................................................................................37 1.3.1 The BRCT Domain.........................................................................................................40 1.4. OBJECTIVES.....................................................................................................................................41 iv CHAPTER 2: REGULATION OF RAD53 THROUGH MODULAR PROTEIN-PROTEIN INTERACTIONS AND MULTI-STEP PHOSPHORYLATION 2.0. INTRODUCTION.............................................................................................................................. 43 2.1. RESULTS……………………………………………………………………………….................46 2.1.0 FHA domains and DNA damage dependent phosphorylation of Rad53………...... 46 2.1.1 Phosphorylation of Rad53 in response to 4-Nitroquinoline Oxide (4-NQO) ……... 59 2.1.2 Phosphorylation of Rad53 in response to Hydroxurea (HU)………………............. 53 2.1.3 Multisite transphosphorylation is required for Rad53 function …............................ 55 2.1.4 Rad9 stimulates Mec1 phosphryaltion of Rad53 in vitro ………………………...... 57 2.1.5 In vitro Rad53 phosphylation sites overlap with those observed in vivo ……......... 63 2.1.6 Class II phosphorylation is required for Rad53 activity……………………………. 65 2.2. DISCUSSION……………………………………………………………………………............... 69 2.2.0 Phosphorylation of Rad53 in response to different genotoxic insults…………….... 69 2.2.1 PIKK-dependent activation of Rad53 orthologs………………………………......... 70 2.2.2 BRCT domain protein, molecular adaptors to the DNA damage-signaling cascade..71 2.2.2.0 Rad9 as a molecular adaptor 2.2.2.1 Mrc1, a potential molecular adaptor for S-Phase specific Rad53 activation 2.2.2.2 Checkpoint Mediators as PIKK adaptors 2.2.3 Regulation of Rad53 off-rate via catalytic autophosphorylation…………………... 78 2.3. TABLES.……………………………………………………………………………..................... 80 2.4. EXPERIMENTAL PROCEDURES…………………………………………………………................ 85 CHAPTER 3: CDC5 REGULATION OF DNA DAMAGE ADAPTATION THROUGH INHIBITION OF RAD53 ACTIVITY 3.0. INTRODUCTION………………………………………………………………………….............. 95 3.1. RESULTS………………………………………………………………………………................ 97 3.1.0 Overexpression of CDC5 leads to inactivation of Rad53 ……………………......... 97 3.1.1 CDC5 phosphoryalte Rad53 in vitro ………………................................................. 100 3.1.2 Rad53 interacts with CDC5 in an FHA-dependent manner ………………….........
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