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Identification of Novel Dna Damage Response Genes Using Functional Genomics

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Identification of Novel Dna Damage Response Genes Using Functional Genomics IDENTIFICATION OF NOVEL DNA DAMAGE RESPONSE GENES USING FUNCTIONAL GENOMICS by Michael Chang A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto © Copyright by Michael Chang (2005) Identification of novel DNA damage response genes using functional genomics Doctor of Philosophy, 2005; Michael Chang; Department of Biochemistry, University of Toronto ABSTRACT The genetic information required for life is stored within molecules of DNA. This DNA is under constant attack as a result of normal cellular metabolic processes, as well as exposure to genotoxic agents. DNA damage left unrepaired can result in mutations that alter the genetic information encoded within DNA. Cells have consequently evolved complex pathways to combat damage to their DNA. Defects in the cellular response to DNA damage can result in genomic instability, a hallmark of cancer cells. Identifying all the components required for this response remains an important step in fully elucidating the molecular mechanisms involved. I used functional genomic approaches to identify genes required for the DNA damage response in Saccharomyces cerevisiae. I conducted a screen to identify genes required for resistance to a DNA damaging agent, methyl methanesulfonate, and identified several poorly characterized genes that are necessary for proper S phase progression in the presence of DNA damage. Among the genes identified, ESC4/RTT107 has since been shown to be essential for the resumption of DNA replication after DNA damage. Using genome-wide genetic interaction screens to identify genes that are required for viability in the absence of MUS81 and MMS4, two genes required for resistance to DNA damage, I helped identify ELG1, deletion of which causes DNA replication defects, genomic instability, and an inability to properly recover from DNA damage during S phase. I also used two-dimensional hierarchical clustering of synthetic genetic interaction data determined by large-scale genetic network analysis to identify RMI1, which encodes a new member of the highly conserved Sgs1-Top3 complex that is an important suppressor of genomic instability. ii ACKNOWLEDGEMENTS Many people contributed to the completion of this thesis. 90% of my gratitude should go to my supervisor, Grant, without whom none of this would be possible. The other 10% goes to others listed below, in no particular order. Brenda Andrews and Craig Smibert: committee members Co-authors: productive and successful scientific collaborations Amy Tong and the Boone lab: help with SGA Charlie Boone: transforming my unsuccessful pombe project into a successful cerevisiae thesis Jiongwen Ou: best lab technician ever Past and present members of the Brown lab: making the journey fun Proceedings of the National Academy of Sciences, USA: authorizing the inclusion of published material in Ch. 3 EMBO Journal: authorizing the inclusion of published material in Ch. 4 and Ch. 5 iii TABLE OF CONTENTS ABSTRACT................................................................................................................................................................... ii ACKNOWLEDGEMENTS ......................................................................................................................................... iii TABLE OF CONTENTS ............................................................................................................................................ iv LIST OF TABLES ....................................................................................................................................................... vi LIST OF FIGURES.................................................................................................................................................... vii LIST OF ABBREVIATIONS ....................................................................................................................................viii PUBLISHED MATERIAL NOT PRESENTED IN THIS THESIS ....................................................................... ix 1. GENERAL INTRODUCTION.................................................................................................................................... 1 1.1 DNA DAMAGE CHECKPOINTS....................................................................................................................... 1 1.2 RecQ DNA HELICASES .................................................................................................................................... 6 1.3 RecQ HELICASES AND TOPOISOMERASE III........................................................................................... 9 1.4 RecQ-Top3 AND DNA DAMAGE CHECKPOINT FUNCTIONS .............................................................. 10 1.5 OTHER PROTEINS IMPORTANT FOR PROCESSING STALLED FORKS ........................................ 12 1.6 ROLE OF HOMOLOGOUS RECOMBINATION IN RESTARTING STALLED FORKS ...................... 15 1.7 YEAST FUNCTIONAL GENOMICS............................................................................................................... 18 1.8 RATIONALE FOR THESIS PROJECT.......................................................................................................... 20 2. MATERIALS AND METHODS............................................................................................................................... 21 2.1 YEAST STRAINS AND MEDIA....................................................................................................................... 21 2.2 HIGH-THROUGHPUT MMS SCREEN ......................................................................................................... 24 2.3 MMS, HU, AND UV SENSITIVITY MEASUREMENTS ............................................................................. 24 2.4 CELL CYCLE SYNCHRONIZATION............................................................................................................. 25 2.5 FLOW CYTOMETRY ........................................................................................................................................ 25 2.6 SYNTHETIC GENETIC ARRAY (SGA) ANALYSIS ................................................................................... 25 2.7 EPITOPE TAGGING, IMMUNOPRECIPITATION, IMMUNOBLOTTING, AND GEL FILTRATION ....................................................................................................................................................................................... 26 2.8 PLASMID LOSS, FORWARD MUTATION RATE, AND Canr MUTATION SPECTRA ....................... 26 2.9 SGA MAPPING (SGAM) ANALYSIS............................................................................................................. 27 2.10 FLUORESCENT MICROSCOPY................................................................................................................. 28 2.11 RECOMBINATION AND GCR ASSAYS .................................................................................................... 28 2.12 PROTEIN HOMOLOGY SEARCHES ......................................................................................................... 28 3. A GENOME-WIDE SCREEN FOR METHYL METHANESULFONATE SENSITIVE MUTANTS REVEALS GENES REQUIRED FOR S PHASE PROGRESSION IN THE PRESENCE OF DNA DAMAGE.......................................................................................................................................................................... 30 3.1 SUMMARY.......................................................................................................................................................... 31 3.2 INTRODUCTION ............................................................................................................................................... 31 3.3 RESULTS AND DISCUSSION........................................................................................................................ 33 4. Elg1 FORMS AN ALTERNATIVE RFC COMPLEX IMPORTANT FOR DNA REPLICATION AND GENOME INTEGRITY................................................................................................................................................... 46 4.1 SUMMARY.......................................................................................................................................................... 47 4.2 INTRODUCTION ............................................................................................................................................... 47 4.3 RESULTS............................................................................................................................................................ 49 4.4 DISCUSSION ..................................................................................................................................................... 67 5. RMI1/NCE4, A SUPPRESSOR OF GENOME INSTABILITY, ENCODES A MEMBER OF THE RecQ HELICASE/TopoIII COMPLEX ................................................................................................................................... 72 5.1 SUMMARY.......................................................................................................................................................... 73 5.2 INTRODUCTION ............................................................................................................................................... 73 5.3 RESULTS...........................................................................................................................................................
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