Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2010 Characterization of Histone H2A Functional Domains Important for Regulation of the DNA Damage Response Elizabeta Gjoneska Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Gjoneska, Elizabeta, "Characterization of Histone H2A Functional Domains Important for Regulation of the DNA Damage Response" (2010). Student Theses and Dissertations. Paper 267. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. CHARACTERIZATION OF HISTONE H2A FUNCTIONAL DOMAINS IMPORTANT FOR REGULATION OF THE DNA DAMAGE RESPONSE A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Elizabeta Gjoneska June 2010 © Copyright by Elizabeta Gjoneska 2010 CHARACTERIZATION OF HISTONE H2A DOMAINS IMPORTANT FOR REGULATION OF THE DNA DAMAGE RESPONSE Elizabeta Gjoneska, Ph.D. The Rockefeller University 2010 DNA double strand breaks represent deleterious lesions which can either be caused by environmental or endogenous sources of DNA damage. Efficient DNA damage response which ensures repair of these lesions is therefore critical for maintenance of genomic stability. The repair happens in the context of chromatin, a three-dimensional nucleoprotein complex consisting of DNA, histones and associated proteins. As such, mechanisms that modulate chromatin structure, many of which involve the histone component of chromatin, have been shown to play a role in regulation of the DNA damage response. In my thesis work I characterize two conserved histone H2A functional domains that are required for normal response to DNA damage. In the first part of my thesis, my collaborators and I demonstrate that Tetrahymena major histone H2A.S contains an H2A.X variant-specific SQ motif within its C-terminal tail, providing the first description of this region in ciliated protozoa. The function of the SQ motif is mediated by post-translational phosphorylation of the conserved serine which is essential for normal progression through Tetrahymena life cycle, and in particular, meiosis. This study provides the first evidence for the existence of meiotic DSBs in Tetrahymena and defines the time interval of meiotic recombination in this organism. In the second part of my thesis, I describe a functional domain which encodes a unique and previously unrecognized role for the histone H2A N- terminal tail in the DNA damage response in S. cerevisiae. A DNA damage survival property exists within the conserved SRS motif spanning residues 17-19 of a single turn α-helical region in the H2A tail, known as the ‘knuckle’. I demonstrate that the SRS motif is required for efficient checkpoint recovery following successful repair, a function independent of post-translational modifications. Another contribution of histone H2A in S. cerevisiae, specific to the MMS- induced DNA damage response, is provided by the three amino-terminal lysines which appear to be functionally redundant. My collaborators and I demonstrate that in vivo two of the lysines, H2A K4 and H2A K7, are acetylated individually as well as together, and identify the third lysine, H2A K13, as a novel acetylation site in S. cerevisiae. ACKNOWLEDGEMENTS iii TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………...…iii TABLE OF CONTENTS………………………………………………………………iv LIST OF FIGURES……………………………………………………………….......viii LIST OF TABLES………………………………………………………………………xi LIST OF ABBREVIATIONS …………………………………………………..….xii CHAPTER 1: GENERAL INTRODUCTION…………………………………….. 1 The nucleosome……………………………………………………….………..2 Histones………………………………………………………………………….4 The higher order structure…………………………………………………….5 DNA damage……………………………………………………………………8 Chromatin structure dynamics………………………………………………11 Histone variants………………………………………………………..12 H2A.X…………………………………………………………...13 Post-translational modifications of histones………………………...15 Phosphorylation………………………………………………..19 Acetylation……………………………………………………...22 Methylation……………………………………………………..24 Ubiquitylation…………………………………………………..26 Recruitment of effector proteins……………………………………...26 ATP-dependent chromatin remodeling……………………………...30 Meiosis………………………………………………………………………….33 Tetrahymena thermophila…………………………………………………….36 Tetrahymena Life cycle……………………………………….………..37 Tetrahymena as a model organism……………………………….........42 iv CHAPTER 2: PHOSPHORYLATION OF HISTONE H2A.S AT THE SQ MOTIF IS REQUIRED FOR DNA REPAIR AND MEIOSIS IN TETRAHYMENA THERMOPHILA………………………………………………….45 Introduction………………………………………………………………………..45 Results………………………………………………………………………............48 Tetrahymena thermophila H2A.S is phosphorylated in response to induced DSBs……………………………………………………………………………..48 Tetrahymena thermophila H2A.S is phosphorylated during meiosis……….49 H2A.S is phosphorylated in developing macronuclei undergoing DNA rearrangement, but not during programmed nuclear death of parental macronuclei..…………………………………………………………................53 H2A.S phosphorylation occurs on the C-terminal S134……………………54 Absence of H2A.S S134 phosphorylaton leads to meiotic defects………...58 Discussion………………………………………………………………………….61 CHAPTER 3: THE AMINO-TERMINAL SRS MOTIF OF SACCHAROMYCES CEREVISIAE HISTONE H2A IS IMPORTANT FOR PROPER DNA DAMAGE RESPONSE……………………………………………………………………………..65 Introduction………………………………………………………………………..65 Results………………………………………………………………………………68 Deletion of the amino-terminal tail of S. cerevisiae histone H2A confers sensitivity to DNA damaging agents………………………………………...68 Histone H2A N-terminal acetylation mapped by mass spectrometry confers subtle sensitivity to MMS…………………………………………….71 The DNA damage-survival property of histone H2A amino-terminal tail is encoded in the ‘knuckle’ region………………………………………………76 Expression of wild type histone H2A suppresses damage sensitivity of strains containing ‘knuckle’ point mutations………….……………………80 v Discussion……………………………………………………………………………...83 CHAPTER 4: FUNCTIONAL ANALYSIS OF HISTONE H2A ‘KNUCKLE’ REGION………………………………………………………………………………...90 Introduction………………………………………………………………………..90 Results………………………………………………………………………………93 Damage sensitivity to HU conferred by ‘knuckle’ S17E mutation is suppressed by overexpression of genes involved in cellular metabolism and protein synthesis…………………………………………………………..93 Transcription of several DNA damage response genes is affected by mutations in the ‘knuckle’ region…………………………………………….95 H2A ‘knuckle’ region is required for efficient DSB repair by the NHEJ pathway…………………………………………………………………………97 H2A ‘knuckle’ residues are not required for DNA DSB break repair using a homologous single strand annealing mechanism……………………….100 Mutations in the H2A ‘knuckle’ region have checkpoint termination defects………………………………….……………………………………....104 Discussion…………………………………………………………………………….108 CHAPTER 5: GENERAL DISCUSSION…………………………………………112 Carboxy-terminal SQ domain of Tetrahymena histone H2A.S…………….112 Is γH2A.X required for efficient DSB repair in Tetrahymena?.....................113 Is the DSB repair function of γH2A.X required for meiosis in Tetrahymena? ………..…………………………………………………………………………114 Amino-terminal ‘knuckle’ domain of S. cerevisiae histone H2A………….117 Is nucleosome structure affected by ‘knuckle’ mutations?.........................120 Transcriptional regulation - is expression checkpoint recovery genes affected by ‘knuckle’ structure?......................................................................121 Is chromatin assembly after repair affected by ‘knuckle’ structure? ……122 vi Reassembly versus restoration - is reinstatement of histone modifications after repair affected by ‘knuckle’ structure perturbations?........................124 How is chromatin reassembly or restoration regulated by ‘knuckle’ structure?............................................................................................................126 CHAPTER 6: MATERIALS AND METHODS…………………………………..128 APPENDIX……………………………………………………………………………145 REFERENCES………………………………………………………………………...147 vii LIST OF FIGURES Figure 1.1: Histone H2A ‘knuckle’ localization within the NCP structure……..3 Figure 1.2: DNA compaction into higher-order chromatin structure…………...6 Figure 1.3: Pathways for DNA double strand break repair…………………….11 Figure 1.4: Post-translational modifications of core histones…………………...17 Figure 1.5: Molecular mechanisms of histone modifications…………………...18 Figure 1.6: Ciliate life cycle…………………………………………………………40 Figure 2.1: Double strand break (DSB) - induced phosphorylation of the conserved C-terminal SQ motif in Tetrahymena thermophila detected by anti γH2A.X specific antibody…………………………………….49 Figure 2.2: Anti- γH2A.X antibody detects meiotic DSB phosphorylation in Tetrahymena thermophila………………………………………………..52 Figure 2.3: Tetrahymena histone H2A.S is phosphorylated in developing macronuclei undergoing DNA rearrangement, but not during programmed nuclear death of parental macronuclei…..…………..54 Figure 2.4: Histone H2A.S C-terminal S134 is the substrate for γH2A.X- detected DSB-induced phosphorylation in Tetrahymena…………...56 Figure 2.5: Disruption of S134 phosphorylation leads to premature termination of conjugation after meiosis II………………………………………...59 Figure 3.1: S. cerevisiae H2A-H2B histone plasmid-shuffle strain……………...69