University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School November 2017 The Role of Sgs1 and Exo1 in the Maintenance of Genome Stability. Lillian Campos-Doerfler University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the Cell Biology Commons, and the Molecular Biology Commons Scholar Commons Citation Campos-Doerfler, Lillian, "The Role of Sgs1 and Exo1 in the Maintenance of Genome Stability." (2017). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/7006 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. The Role of Sgs1 and Exo1 in the Maintenance of Genome Stability By Lillian Campos-Doerfler A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy With a concentration in Cell and Molecular Biology Department of Cell Biology, Microbiology and Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Kristina Schmidt, Ph.D. Meera Nanjundan, Ph.D. Stanley Stevens, Ph.D. Sandy Westerheide, Ph.D. Date of Approval: November 17, 2017 Keywords: Genome Instability, DNA repair, RecQ Helicases, Sgs1, Exo1 Copyright 2017, Lillian Campos-Doerfler Dedication I would like to dedicate this dissertation to my amazing and supportive family. To my husband Eric for his constant encouragement and patience, my sister Connie for her motivational phone calls and guidance, and my parents Wanda and Jaime for everything they have done that allowed for me to pursue my dream. Acknowledgements I would first like to acknowledge my advisor Dr. Kristina Schmidt for the opportunity and privilege to study under her expertise. I thank my committee members for their time and input that helped guide my studies. To current and former graduate students that shared in my triumphs and failures, always helping me take both in stride. I would like to thank our former post-doctoral fellow Lorena for encouraging me when I was just a technician to believe in myself enough to take the leap and apply to graduate school. I appreciate the help and guidance of all those mentioned that contributed to my success. Table of Contents List of Tables ……………………………………………………………………………... ........ vi List of Figures…………………………………………………………………………...... ...... viii Abstract.................................................................................................................... ........ xi Chapter One: Introduction........................................................................................ ........ 1 Human Disease Linked to Defects in the RecQ helicases............................ ........ 2 Bloom’s Syndrome............................................................................. ........ 2 Werner Syndrome.............................................................................. ........ 5 Rothmund Thomson Syndrome......................................................... ........ 8 RecQ Family of Helicases............................................................................. ...... 10 RecQ-like Helicase Sgs1 in Saccharomyces cerevisiae............................... ...... 13 Sgs1 Substrates and Physical Interaction Partners...................................... ...... 13 DNA Double-Strand Break Repair................................................................ ...... 18 Common Early Steps of HR Repair of DNA Double-strand Breaks.............. ...... 18 HR Directed Repair Following Sgs1 and Exo1 Resection of DNA Ends....... ...... 21 The Role of Sgs1 at Stalled Replication Forks.............................................. ...... 22 The Role of Sgs1 in Meiosis......................................................................... ...... 25 DNA Repair Exonuclease, Exo1................................................................... ...... 26 Functions of Exo1 in Mismatch Repair............................................... ...... 26 Regulation of Exo1 in Response to DNA Damage............................. ...... 27 Hypothesis and Aims.................................................................................... ...... 28 Significance................................................................................................... ...... 39 i References.................................................................................................... ...... 30 Chapter Two: Differential Genetic Interactions Between Sgs1, DNA-damage Checkpoint Components and DNA Repair Factors in the Maintenance of Chromosome Stability........................................................................................ ...... 56 Abstract......................................................................................................... ...... 56 Introduction................................................................................................... ...... 58 Results and Discussion................................................................................. ...... 60 Functional interaction between Sgs1 and DNA-damage checkpoint components Mec3, Mec1, Tel1, Dun1 and Chk1 in the suppression of chromosomal translocations................................. ...... 60 Deletion of RAD59 inhibits spontaneous interchromosomal translocations between short repeats........................................... ...... 64 Candidate screen reveals EXO1 as a strong suppressor of GCR formation in cells lacking Sgs1.................................................... ...... 68 Conclusion.................................................................................................... ...... 72 Methods........................................................................................................ ...... 73 Yeast strains and media..................................................................... ...... 73 Sensitivity to DNA damaging agents HU and MMS........................... ...... 73 Flucuation assays............................................................................... ...... 74 Protein extraction and Western blot analysis..................................... ...... 75 Figures and tables......................................................................................... ...... 76 References.................................................................................................... ...... 85 Chapter Three: Exo1 phosphorylation status controls the hydroxyurea sensitivity of cells lacking the Pol32 subunit of DNA polymerases delta and zeta.............. ...... 96 Abstract......................................................................................................... ...... 96 Introduction................................................................................................... ...... 97 Materials and methods.................................................................................. .... 101 Yeast strains and media.............................................................. .... 101 Gross-chromosomal rearrangement (GCR) assay...................... .... 102 Fluctuation assays and CAN1 mutation spectrum analysis......... .... 102 ii Hydroxyurea (HU) and methylmethanesulfonate (MMS) sensitivity assays........................................................................................... .... 103 Results.......................................................................................................... .... 103 Functional interactions between Exo1, DNA repair and DNA- damage response........................................................................ .... 103 Phosphorylation-site mutants of Exo1 are hypermorphic in HU- treated pol32Δ cells..................................................................... .... 105 Mismatch repair activity partially contributes to Exo1 toxicity in pol32Δ cells ................................................................................ .... 108 Discussion ..................................................................................................... .... 111 Figures and tables......................................................................................... .... 117 References.................................................................................................... .... 134 Chapter Four: Sgs1 binding to Rad51 stimulates homology-dependent DNA repair in Saccharomyces cerevisiae............................................ .... 146 Abstract......................................................................................................... .... 146 Introduction................................................................................................... .... 147 Materials and methods.................................................................................. .... 150 Yeast strain and media....................................................................... .... 150 DNA-damage sensitivity assays......................................................... .... 151 Gross-Chromosomal Rearrangement (GCR) Assay.......................... .... 151 Mutator Assays and Mutation Spectrum Analysis.............................. .... 152 Tetrad analysis................................................................................... .... 152 Pull-Down Assay and Western Blots.................................................