Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2011 Role of Histone Gene Dosage in DNA Damage Repair Dun Liang Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF MEDICINE ROLE OF HISTONE GENE DOSAGE IN DNA DAMAGE REPAIR By DUN LIANG A dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Summer Semester, 2011 The members of the committee approve the Dissertation of Dun Liang defended on July 1st, 2011. __________________________________ Akash Gunjan Professor Directing Dissertation __________________________________ David Gilbert University Representative __________________________________ Myra Hurt Committee Member __________________________________ Johanna Paik Committee Member __________________________________ Yanchang Wang Committee Member Approved: _____________________________________ Richard Nowakowski, Chair, Department of Biomedical Sciences _____________________________________ John Fogarty, Dean, College of Medicine The Graduate School has verified and approved the above-named committee members. ii To be as firm and resolute in my actions as I was able, and not to adhere less steadfastly to the most doubtful opinions, when once adopted, than if they had been highly certain… To endeavor always to conquer myself rather than fortune, and change my desires rather than the order of the world, and in general, accustom myself to the persuasion that, except our own thoughts, there is nothing absolutely in our power; so that when we have done our best in things external to us, all wherein we fail of success is to be held, as regards us, absolutely impossible… --Discourse on the Method, René Descartes, 1637 iii ACKNOWLEDGEMENTS My PhD training is an odyssey both in science and life. I am full of gratitude to people who helped me all the way along. First and foremost I would like to thank my advisor Dr. Akash Gunjan, who is not only a good teacher but also a good friend to me, for his immeasurable support during the past six years. He taught me to do science, to think like a scientist, and most importantly, to live an exploring, creative and productive life. He has always been generous towards my confusions and mistakes; he provided me with freedom and space for development, and with prompt guidance or directions when needed; he showed me the appropriate bearing towards daily frustration and disappointments. He is a true role model in motivating me to pursue in science. I would not be who I am today without his mentoring. Secondly, I would like to acknowledge my other committee members: Drs. Myra Hurt, Yanchang Wang, David Gilbert and Johanna Paik. They offered me invaluable support, encouragement and advice towards my research. I deeply appreciate the extra effort of Dr. Wang put in to help me to adapt to the cultural differences as a foreign student and provide me with many yeast strains and other materials. Special thanks are due to Dr. Hurt for her consistent financial support and understanding. She will be remembered by my family as serving on the PhD committees for both my wife and me. Additionally thanks to Dr. Gilbert, who has always been the source of insight about the beauty of science and to Dr. Paik, who provided me countless support and care. I take this opportunity to thank them sincerely and deeply. As a member of Gunjan lab family, I express my appreciation to my lab colleagues. It has been a great pleasure to work and share my life with them in the lab. iv Special acknowledgement goes to Dr. Rakesh Singh, who helped to open my eyes and mind in science, as well as to provide critical inputs for my projects. He will be also thanked for his persistence in asking all those “forbidden to graduate students” questions to me over the last few years to keep me awake. Many thanks due to Ugander Reddy for the help in optimizing my experiments and for being a great companion in the lab. I would like to give a special thank to Marie-Helene Kabbaj for her endless support in lab management and material preparation. Apart from the work, I also learned to be a healthy and eco-friendly scientist from them all. Here within, I would like to acknowledge other faculties, staffs, postdocs and graduate students in the Department of Biomedical Sciences, who have created such a friendly, dynamic, supportive and inspiring research environment. For those not specifically mentioned, please accept my sincerest appreciation for your help and assistance. I am also filled with gratitude to my parents for providing me the best education opportunities available and understanding of the path that I chose in my life. Their love and support granted me the opportunity to move forward without any hesitation or concern. Finally, words of thanks are due to my wife, who I met, fell in love with and married here in FSU. She makes my life different, she makes me home. I could not finish this work without her company and comfort. Thank you. v TABLE OF CONTENTS List of Tables...................................................................................................................ix List of Figures.................................................................................................................. x Abstract ..........................................................................................................................xii 1. DNA DAMAGE AND REPAIR.................................................................................. 1 1.1 Various Types of DNA Damage ...................................................................... 1 1.2 Different Types of DNA Damage Repair ......................................................... 4 1.2.1 Direct repair.......................................................................................... 5 1.2.2 Mismatch repair.................................................................................... 6 1.2.3 Excision repair...................................................................................... 6 1.2.4 Break repair....................................................................................................10 1.3 Choice of DSB repair pathway: HR vs. NHEJ ............................................... 26 1.3.1 Nature of DNA DSB breaks ................................................................ 27 1.3.2 Effect of cell cycle regulation on the choice of DSB repair pathway ... 29 1.3.3 NHEJ factor’s binding to the break ends ............................................ 30 1.3.4 DSB repair pathway choice in different species ................................. 31 1.4 DNA Damage Signaling in Budding Yeast .................................................... 33 2. THE ROLE OF HISTONES, CHROMATIN STRUCTURE, AND EPIGENETIC MODIFICATIONS IN DNA REPAIR .............................................................................. 38 2.1 Histones Serve as Primary Protein Components of Chromatin..................... 38 2.2 Definition of Epigenetics................................................................................ 42 2.3 Overview of Histone Modifications as Epigenetic Marker.............................. 43 2.4 Role of Histone Modifications in DSB Repair ................................................ 45 2.4.1 H2A phosphorylation .......................................................................... 45 2.4.2 Histone ubiquitination ......................................................................... 46 2.4.3 Methylation of H3K79 and H4K20 ...................................................... 48 2.4.4 Histone acetylation ............................................................................. 49 2.5 Chromatin Remodeling and DNA Repair ...................................................... 52 2.6 Role of Histone Chaperones in DSB Repair.................................................. 58 2.7 DNA methylation and DNA damage repair.................................................... 62 3. HISTONES: THE ENEMY WITHIN........................................................................ 64 3.1 Histones: Enemy or Friend?.......................................................................... 64 3.2 Sources of Excess Histones.......................................................................... 68 3.2.1 Multiple histone genes........................................................................ 69 3.2.2 Replication arrest................................................................................ 72 3.2.3 Histones evicted during transcription or DNA damage repair ............. 73 3.3 Regulation of Histone Level .......................................................................... 75 3.3.1 Transcriptional control of histone gene expression ............................ 75 vi 3.3.2 Posttranscriptional regulation of histone gene expression.................. 78 3.3.3 Posttranslational regulation of histone proteins .................................. 79 3.3.4 Histone chaperones in regulating histone proteins............................. 80 4. EXCESS HISTONE LEVELS MEDIATE CYTOTOXICITY VIA MULTIPLE MECHANISMS.............................................................................................................. 83 4.1 Abstract......................................................................................................... 83 4.2 Introduction ..................................................................................................