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Barbour-Thesis.Pdf (2.004Mb) Synthetically Lethal Interactions Classify Novel Genes in Postreplication Repair in Saccharomyces cerevisiae A thesis submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Microbiology and Immunology University of Saskatchewan Leslie Barbour, B.Sc. © Copyright Leslie Barbour, February 2005. All rights reserved. Permission to Use In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or make other use of material in this thesis in whole or in part should be addressed to: Head of the Department of Microbiology and Immunology Health Sciences Building, 107 Wiggins Road University of Saskatchewan Saskatoon, SK Canada S7N 5E5 i Acknowledgments First I would like to thank my supervisor, Dr. Wei Xiao for providing a supportive learning environment and invaluable guidance during my research project. His devotion and enthusiasm towards science and patience and encouragement has been a motivating force throughout my studies. I would also like to thank members of my supervisory committee, Dr. Sean Hemmingsen, Dr. Harry Deneer, Dr. Troy Harkness and Dr. Gerry Rank for offering support and advice. A special thanks to Dr. Hannah Klein of NYU Medical Center for accepting to serve as my external examiner. I would like to thank current and previous members of Dr. Xiao’s laboratory: Parker Anderson, Lyn Ashley, Yu Fu, Xuming Jia, Katherine Lockhart, Michelle Hanna, Landon Pastushok, Noor Syed, Lindsay Ball, XinFeng Ma, Rui Wen, Stacey Broomfield, Yu Zhu, Todd Hryciw, Barbara Chow and Treena Swanston for their kindness and making the workplace fun. I would like to thank the Department of Microbiology and Immunology, College of Medicine and Dr. Wei Xiao for their financial support. Finally, I would like to thank my family for their support and patience during my studies. ii Abstract Both prokaryotic and eukaryotic cells are equipped with DNA repair mechanisms to protect the integrity of their genome in case of DNA damage. In the eukaryotic organism Saccharomyces cerevisiae, MMS2 encodes a ubiquitin-conjugating enzyme variant protein belonging to the RAD6 repair pathway; MMS2 functions in error-free postreplication repair (PRR), a subpathway parallel to REV3 mutagenesis. A mutation in MMS2 does not result in extreme sensitivity to DNA damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. By taking advantage of the synergism between error-free PRR and mutagenesis pathway mutations, a conditional synthetic lethal screen was used to identify novel genes genetically involved in PRR. A synthetic lethal screen was modified to use extremely low doses of MMS that would not affect the growth of single mutants, but would effectively kill the double mutants. Fifteen potential mutants were characterized, of which twelve were identified as known error-prone PRR genes. Characterization of mutations in strains SLM-9 and SLM-11, that are conditionally synthetically lethal with mms2∆, revealed functions for both checkpoints and mating-type heterozygosity in regulating PRR. Cell cycle checkpoints monitor the integrity of the genome and ensure that cell cycle progression is deferred until chromosome damage is repaired. The checkpoint genes genetically interact with both the error-free and error-prone branches of PRR, potentially for delaying cell cycle progression to allow time for DNA repair, and for signaling the stage of the cell cycle and thus DNA content. Other potential monitors for DNA content are the a1 and α2 proteins encoded by the mating type genes iii MATa and MATα, respectively. Diploid cells heterozygous for mating type (a/α) show an increased resistance to UV damage and are more recombination-proficient than haploid cells. Haploid PRR mutants expressing both mating type genes show an increased resistance to DNA-damaging agents. This phenomenon is specific to PRR: it was not seen in excision repair-deficient and recombination-deficient mutants tested. The rescuing effect seen in PRR mutants heterozygous for mating type is likely the result of channeling lesions into a recombination repair pathway and away from the non-operational PRR pathway. Both checkpoint and mating type genes play a role in regulating PRR. Almost certainly these genes are required to monitor the cell cycle stage and DNA content to determine the best mechanism to repair the damaged DNA thus preventing genomic instability. iv Table of Contents Permission to Use............................................................................................................. i Acknowledgments........................................................................................................... ii Abstract ..........................................................................................................................iii Table of Contents............................................................................................................ v List of Tables.................................................................................................................. ix List of Figures ................................................................................................................. x List of Abbreviations..................................................................................................... xi Chapter One -- Introduction ......................................................................................... 1 1.1. DNA Damage ....................................................................................................... 1 1.2. Types of DNA Damage........................................................................................ 1 1.2.1. Spontaneous DNA Damage.......................................................................... 2 1.2.2. DNA Polymerase Proofreading Prevents Copying Errors ....................... 4 1.2.3. Exogenous DNA Damage............................................................................. 5 1.2.3.1. Physical DNA Damaging Agents.......................................................... 5 1.2.3.2. Chemical DNA Damaging Agents........................................................ 6 1.3. Repair of DNA Damage ...................................................................................... 8 1.3.1 Reversal of Damage....................................................................................... 9 1.3.2. Excision Repair........................................................................................... 11 1.3.3. Mismatch Repair ........................................................................................ 14 1.3.4. DNA Damage Tolerance ............................................................................ 16 1.3.5. Repair of Strand Breaks ............................................................................ 16 1.3.5.1. Mechanisms of Homologous DSB Repair.......................................... 18 1.3.5.2. DSB Repair Model............................................................................... 18 1.3.5.3. Gene Conversion.................................................................................. 19 1.3.5.4. The Synthesis-Dependent Strand Annealing Model ........................ 21 1.3.5.5. Break-Induced Replication................................................................. 21 1.3.5.6. Single Strand Annealing ..................................................................... 23 1.3.5.7. Genes Involved in Recombination Repair......................................... 23 1.3.5.8. Non-homologous End-joining............................................................. 25 1.4. Postreplication Repair in Prokaryotes ............................................................ 27 1.4.1. SOS Response in E. coli.............................................................................. 29 1.4.2. Recombination-mediated Replication Fork Restart ............................... 30 1.5. Post-replication Repair in Saccharomyces cerevisiae ..................................... 31 1.5.1. Recombination Repair in DNA Damage Tolerance ................................ 31 1.5.1.1. Recombination-mediated Replication Fork Restart in Eukaryotes 31 1.5.1.2. Structure-specific Endonucleases in Eukaryotes.............................. 35 1.5.2. Error-free and Error-prone Replication Fork Bypass ........................... 37 1.5.2.1. Error-prone Replication Fork Bypass............................................... 39 1.5.2.2. Error-free Replication Fork Bypass .................................................. 40 1.5.3. Role of Srs2 in Replication Bypass...........................................................
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