UHRF1 and the DNA damage response by Helena Sumantrai Mistry A thesis submitted in conformity with the requirements For the Degree of Doctor of Philosophy Graduate Department of Pharmacology & Toxicology University of Toronto ©Copyright by Helena Sumantrai Mistry 2011 UHRF1 and the DNA damage response Helena Sumantrai Mistry Doctor of Philosophy, 2011 Graduate Department of Pharmacology & Toxicology University of Toronto THESIS ABSTRACT Our DNA is under constant threat from endogenous and exogenous damaging agents. Our cells have evolved a network of signaling pathways and repair mechanisms that detect and counteract this threat, collectively referred to as the DNA damage response. Cells that lose the ability to cope with DNA damage risk the acquisition of deleterious changes to DNA sequence or structure. I initially set out to identify and characterize candidate proteins that interact with Mus81- Eme1, an endonuclease that processes DNA intermediates that arise from aberrant or stalled DNA replication. I focused on one interesting candidate protein known as Nuclear protein 95 (Np95) which now is called UHRF1 (Ubiquitin-like, containing PHD and RING finger domains, 1). Although previous studies demonstrate the importance of Mus81-Eme1 enzyme in DNA repair, genome integrity, and tumor suppression, little is known about how the enzyme acts together with other components of signaling pathways that comprise the DNA damage response. My findings in chapter two characterized this interaction and linked Mus81-Eme1 with UHRF1 in the cellular response to DNA damage. Although UHRF1 levels have been linked with sensitivity to antineoplastic agents, a direct role for UHRF1 in the DNA damage response had not been elucidated or reported. Accordingly, the third chapter of my thesis focuses on investigating the role of UHRF1 in the cellular response to DNA damage caused by exposure to γ-irradiation. Our findings for chapter three indicate that (i) UHRF1 is crucial for the cellular ii response to double strand breaks caused by γ-irradiation and that (ii) UHRF1 is critical for maintenance of chromosome integrity. Recent studies have now implicated UHRF1 in processes required for heterochromatin replication. This protein has been shown to play a role in the replication of heterochromatin by helping to replicate DNA methylation patterns and playing a role in propagating the epigenetic mark known as histone 3 lysine 9 trimethylation (H3K9me3). H3K9me3 has been shown to play a role in a signaling pathway involved in the repair of DNA damage in heterochromatic regions. In the fourth chapter of my thesis, we hypothesize that UHRF1 is playing a role in a pathway that responds to DSB damage in heterochromatic regions of chromatin. Our results indicate that a loss of UHRF1 results in a loss of heterochromatic H3K9me3 and heterochromatin associated HP1β. Our findings support the idea that epigenetic alterations maintained by UHRF1 contribute to signals that relax heterochromatin to facilitate access for repair factors. In summary, findings presented in this thesis shed light on processes that protect cells from DNA damage caused by radiation and chemotherapy and safeguard genome integrity. iii ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Peter McPherson for giving me the opportunity to pursue research in his laboratory. I am grateful for his mentorship and belief in my ability as a scientist. His leadership skills, enthusiasm and encouragement will forever be admired and never be forgotten. His dedication to the laboratory and ability to put down whatever he is working on, and listen to my daily experimental update is greatly appreciated. I would also like to thank Dr. Patricia Harper and Dr. Jason Matthews for their constructive comments during my committee meetings. I owe a special thanks to Dr. Jane Mitchell and Dr. Denis Grant for the career advice and encouragement along the way. I am grateful to past and present members of the laboratory: Haya Sarras, Brenda Yun, Loni Gibson, Solmaz Alizadeh, Meghan Larin, Hussein Butt, Aileen Gracias and Daniel Sisgoreo. A special thanks to Laura Tamblyn for the help with FACS analysis and mentorship throughout the program. I would like to also thank my friends in the department for their encouragement and support. A special mention goes to Rosalia Yoon, Aman Mann, Shuang Wang (Sammi), Lick Lai, Ewa Hoffman and Craig Hayden for all their support. My hard work and experimental dedication does not compare to the encouragement and consistent support I have received from my mother. During the good times and the tougher times, she has always supported me emotionally and financially. Words cannot express my gratitude towards her never ending support, and guidance in making my dream a reality. I know I have been very difficult at times, and the patience she has displayed as well as her love and iv support have allowed me to achieve my career goals. Dear mother, I could not have done this without you. v TABLE OF CONTENTS THESIS ABSTRACT ................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................ iv TABLE OF CONTENTS ............................................................................................................ vi LIST OF PUBLICATIONS ......................................................................................................... x LIST OF ORAL AND POSTER PRESENTATIONS ............................................................... x ABBREVIATIONS ..................................................................................................................... xii LIST OF TABLES ..................................................................................................................... xiv LIST OF FIGURES .................................................................................................................... xv CHAPTER ONE: GENERAL INTRODUCTION .................................................................... 1 I.1 Importance of Genome Integrity. ............................................................................................. 1 I.2 DNA damage- characteristic lesions and their sources ............................................................ 3 I.2.1 Endogenous sources ....................................................................................................... 3 I.2.2 Environmental Radiation................................................................................................ 5 I.2.3 Antineoplastic agents ...................................................................................................... 5 I.3 The DNA damage response ....................................................................................................... 6 I.3.2 Surveillance and signalling in response to DNA double-strand break damage .............. 9 I.4 Cellular events triggered by the DNA damage response ........................................................ 11 I.4.1 Cell cycle arrest and senescence ................................................................................... 11 1.4.2 Apoptosis ...................................................................................................................... 12 I. 4.3 DNA repair mechanisms .............................................................................................. 14 I.5 Consequences of impaired DNA damage response and loss of genome integrity .................. 23 I.5.1 Diseases linked with defective responses to DNA damage .......................................... 24 1.5.2 Defective responses to DNA damage and neoplastic transformation .......................... 24 I.5.3 Aging ............................................................................................................................. 26 I.6 Role of Mus81- Eme1 endonuclease in the DNA damage response ....................................... 26 I.6.1 Mus81 and Genome Instability ..................................................................................... 30 vi I.7 The DNA damage response and its relationship to chromatin ................................................ 31 I.7.1 The heterogeneous nature of mammalian chromatin .................................................... 31 1.7.2 Chromatin dynamics/remodelling ................................................................................ 33 I.7.3 Post-translational modifications of histones that demarcate chromatin states .............. 34 I.7.4 Histone Acetylation ....................................................................................................... 34 I.7.5 Histone Lysine Methylation .......................................................................................... 35 I.7.6 Ubiquitination of Histones ............................................................................................ 37 I.7.7 Histone Variant Incorporation ....................................................................................... 37 I.7.8 DNA methylation .......................................................................................................... 39 I.8 Chromatin Accessibility and DNA repair ............................................................................... 40 1.8.1 Unmasking of H3K9me3 facilitates activation of Tip60 Histone Acetyltransferase during the DNA damage response ........................................................................................