Formation of Dicentric and Acentric Chromosomes, by a Template Switch Mechanism, in Budding Yeast Item Type text; Electronic Dissertation Authors Paek, Andrew Luther Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 04/10/2021 02:16:46 Link to Item http://hdl.handle.net/10150/194260 1 FORMATION OF DICENTRIC AND ACENTRIC CHROMOSOMES, BY A TEMPLATE SWITCH MECHANISM, IN BUDDING YEAST By Andrew L. Paek A Dissertation Submitted to the Faculty of the DEPARTMENT OF MOLECULAR AND CELLULAR BIOLOGY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2010 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Andrew Paek Entitled Formation of Dicentric and Acentric Chromosomes, by a Template Switch Mechanism, in Budding Yeast And recommend that it be accepted as fulfilling the dissertation requirement for the degree of Doctor of Philosophy _______________________________________________________________Date: 10/01/2010 Ted Weinert _______________________________________________________________Date: 10/01/2010 Kathleen Dixon _______________________________________________________________Date: 10/01/2010 John Little _______________________________________________________________Date: 10/01/2010 Lisa Nagy _______________________________________________________________Date: 10/01/2010 Roy Parker Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. _______________________________________________________________Date: 10/01/2010 Dissertation Director: Ted Weinert 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Request for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the author Signed: Andrew Paek 4 ACKNOWLEDGMENTS I would like to thank my wonderful family for all the support over the years. To my parents, Don and Carol Paek, thanks for being loving parents and providing a wonderful education. To my siblings, Elizabeth McAshan and Donald Paek, thanks for your friendship and support over the years. I would also like to thank my future wife, Molly Stothert-Maurer, words cannot describe how important your presence has been throughout this whole process. You listened to my talks, you gave me support when needed, you even moved to the desert for me; I am extremely lucky to have a friend like you. I would also like to thank my advisor Ted Weinert. Throughout my graduate career at the University of Arizona, Dr. Weinert has been both an excellent mentor and a friend. Ted was always available, especially if you wanted to discuss science and I will always appreciate that. He has a truly admirable enthusiasm for both science and life that I have always looked up to. I have learned so much from him, and will always be indebted to him for this. I would also like to thank the other members of the Weinert lab. I always felt that working as a team was much more fun and productive than going solo, and the graduate students in the lab were always willing to collaborate with me. To Hope Jones, thanks for all the great science discussions, collaborations and most importantly the pranks. To Salma Kaochar, it was a pleasure working together with you on so many things, your intelligence and friendly personality made the lab a wonderful place to be. To Lisa Shanks, thanks for keeping the lab running and for all the fun conversations over the years. I also thank Arshed Al-Obeidi for being one of the hardest working undergraduates even until the last minute. All the undergraduates in the Weinert lab have added a lot to the group, so I would also like to thank Aly Elezaby, Cody Witham and Joseph Chao. Special thanks goes out to the members of the JMST student group, Jay Konieczka, Tom Hartl, Michael Dellinger, Chris “Stretch” Pappas, Chris Bauer, Matt Callan and Chinedu Nworu. These guys helped out with all my talks, helped read through manuscripts, and threw some great parties. Thanks for making graduate school such a wonderful experience. Finally I would like to thank my committee for always being there for science advice and keeping me on my toes. You have all helped me to grow intellectually and I appreciate that. I would also like to thank Andrew Capaldi, though not on my committee he always gave excellent advice and was a wonderful person to talk science with. 5 DEDICATION To the memory of Justin Sparks, who had such a strange and wonderful impact on all who were lucky enough to know him. 6 TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………………………………………………………09 ABSTRACT……………………………………………..……………………………………………………………………..10 AN EXPLANATION OF THE DISSERTATION FORMAT……………………………………..……………….13 CHAPTER I: INTRODUCTION……….……………………………………………………………………………….14 1.1 EXPLANATION OF THE PROBLEM AND ITS CONTEXT…………..………..……………………14 DNA replication…………………………….…………………………………………………………….14 DNA repeats……..………………………………………………………………………………………….15 Chromosome architecture………………………………..………………………………………….16 1.2 A REVIEW OF THE LITERATURE ON REPLICATION BYPASS PATHWAYS AND REPLICATION ASSOCIATED GENOME REARRANGEMENTS………………………………………….18 The many forms of genome rearrangements………………………………………………………18 General mechanisms underlying genome rearrangement…………………………………..19 The five degrees of freedom in replication fork recovery………………………..………….19 Evidence for the forks five degrees of freedom………………………………………...………..20 DNA polymerase uncoupling……………………..……………………………………………….……….22 Template-Switching…..……………………………………………………………………………….……….23 DSBs and stalled forks….…………………………………………………………………………….….……23 When fork recovery fails; role of sequence homology and colocalization…….…..…24 7 TABLE OF CONTENTS - Continued Possible links between fork recovery failure and genome rearrangements…..…..24 CHAPTER II: PRESENT STUDIES………………………………………………………………………………..29 2.1 A BRIEF OVERVIEW OF WHAT WAS KNOWN…..………………………………………………29 2.2 A BRIEF OVERVIEW OF THE LOGIC AND KEY FINDINGS OF THE CURRENT STUDY……………………………………………………………………………………………………..…..32 The unstable chromosomes are dicentric chromosomes…….………….….32 Dicentric chromosomes form at other inverted repeats in the yeast genome………………………………………………………………………………………………..33 Inverted repeat fusion occurs independent of double strand break repair pathways……………………………………………………………………………………….33 Dicentric chromosome formation decreases in polymerase mutants……………………………………………………………………………….………………….35 2.3 ISOLATION AND ANALYSIS OF ADDITIONAL DICENTRIC CHROMOSOMES…38 2.4 STABILITY OF INVERTED REPEATS IN OTHER GENOMES…………..…………….…..41 2.5 FUSION OF NEARBY INVERTED REPEATS BY A REPLICATION-BASED MECHANISM LEADS TO FORMATION OF DICENTRIC AND ACENTRIC CHROMOSOMES THAT CAUSE GENOME INSTABILITY IN BUDDING YEAST………………………..…………....44 2.6 THE ROLE OF REPLICATION BYPASS PATHWAYS IN DICENTRIC CHROMOSOME FORMATION IN BUDDING YEAST………………………………………………………………..…46 8 TABLE OF CONTENTS - Continued CHAPTER 3: FUTURE STUDIES........................................................................................47 3.1 AN INTRODUCTION TO THE PROBLEM….……………….……………………….………..………47 3.2 HUMANS AND OTHER ORGANISMS HAVE MORE NUANCED REPAIR PATHWAYS…………………………………………………………………………………………...………….48 3.3 STABILIZATION OF ACENTRIC AND DICENTRIC CHROMOSOMES……………………...50 3.4 SUMMARY………………………………………………………………………………………………………..53 REFERENCES………………………………………………………………………………………………………………55 APPENDIX A….…………………………………………………………………………………………………………..68 FUSION OF NEARBY INVERTED REPEATS BY A REPLICATION-BASED MECHANISM LEADS TO FORMATION OF DICENTRIC AND ACENTRIC CHROMOSOMES THAT CAUSE GENOME INSTABILITY IN BUDDING YEAST APPENDIX B……………………………………………………………………………..………………………………135 THE ROLE OF REPLICATION BYPASS PATHWAYS IN DICENTRIC CHROMOSOME FORMATION IN BUDDING YEAST 9 LIST OF FIGURES Figure 1.1: The replication fork’s five degrees of freedom………………………………………….27 Figure 2.1 Chromosome system to detect instability………………………………….…………………31 Figure 2.2 Strategy for the stabilization of dicentric chromosomes………………………………39 Figure 2.3 The frequency of inverted repeats grows exponentially with increasing genome size……………………………………………………………………………….42 10 ABSTRACT Chromosomal rearrangements occur in all organisms and are important both in the evolution of species and in pathology. In this dissertation I show that in Saccharomyces cerevisiae, or budding yeast, one type of chromosomal rearrangement occurs when inverted repeats fuse, likely during DNA replication by a novel mechanism termed “faulty template switching”. This fusion can lead to the formation of either a dicentric or acentric chromosome, depending on the direction of the replication