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The Recognition and Mobility of DNA Double-Strand Breaks The Recognition and Mobility of DNA Double-Strand Breaks by Jonathan Strecker A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto © Copyright by Jonathan Strecker 2016 The Recognition and Mobility of DNA Double-Strand Breaks Jonathan Strecker Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto 2016 Abstract DNA double-strand breaks (DSBs) pose a threat to cell survival and genomic integrity, and remarkable mechanisms exist to deal with these breaks. A single DSB activates a signalling response that profoundly impacts cell physiology, not least through the engagement of DSB repair pathways and the arrest of cell division. Here I study these processes in the budding yeast Saccharomyces cerevisiae and investigate two central themes to this response. First, I examine how the natural ends of chromosomes, telomeres, are differentiated from DSBs by generating DNA ends with increasing telomeric character. I discover a striking transition in the activity of the telomerase inhibitor Pif1 at these ends and propose that this is the dividing line between DSBs and telomeres. Second, I investigate a phenomenon whereby a DSB increases the mobility of chromosomes within the nucleus. This increase in mobility is dependent on the Mec1 kinase and is proposed to promote repair by homologous recombination. I identify that the Mec1-dependent phosphorylation of Cep3, a kinetochore component, is required to stimulate chromatin mobility following DNA breakage and provide a new model for how a DSB affects the constraints on chromosomes. Unexpectedly, I find that increased mobility is not required for DSB repair and instead propose that Cep3 helps arrest the cell cycle in response to a DSB. Finally, my investigation into Cep3 phosphorylation provides new insight into the role of chromatin remodelers and the histone variant H2A.Z in DSB-induced chromatin mobility. ii Acknowledgments I would like to thank my supervisor Daniel Durocher for his mentorship and support, it has been an absolute privilege to begin my journey in such a thriving and successful environment. Many of your lessons and philosophies will undoubtedly remain with me during my career. I thank my committee members Brenda Andrews and Karim Mekhail for their thoughtful comments and suggestions over the years. Finally, I am grateful to members of the Durocher lab past and present, with particular mention to Wei Zhang who taught me the basics of budding yeast and whose results and ideas led to the projects described here. iii Table of Contents Acknowledgments.......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Figures .............................................................................................................................. viii List of Abbreviations ...................................................................................................................... x Chapter 1 Introduction .................................................................................................................... 1 1.1 DNA double-strand breaks threaten genome integrity ........................................................ 1 1.1.1 The causes and consequences of DSBs ........................................................................ 1 1.1.2 Genome instability and cancer development ................................................................ 2 1.2 The DNA double-strand break response .............................................................................. 3 1.2.1 The sensing and signalling of DSBs ............................................................................. 3 1.2.2 Repair of DSBs ............................................................................................................. 6 1.2.3 DSBs arrest cell division ............................................................................................ 12 1.3 DNA ends in the cell .......................................................................................................... 13 1.3.1 The structure and function of telomeres ..................................................................... 13 1.3.2 Telomerase activity and regulation............................................................................. 15 1.3.3 Interplay between DSBs and telomeres ...................................................................... 18 1.3.4 The telomerase inhibitor Pif1 ..................................................................................... 18 1.4 DSB repair in the context of the nucleus ........................................................................... 20 1.4.1 Spatial organization of the nucleus ............................................................................. 20 1.4.2 Nuclear position and genomic integrity...................................................................... 22 1.4.3 Chromatin is not static ................................................................................................ 24 1.4.4 Chromatin mobility following a DSB ........................................................................ 26 1.5 Summary and rationale ...................................................................................................... 27 Chapter 2 Materials and Methods ................................................................................................. 29 2.1 General yeast strains and growth ................................................................................... 29 2.2 Telomere addition strains ............................................................................................... 29 2.3 Chromatin mobility strains ............................................................................................. 30 2.4 Telomere addition analysis ............................................................................................. 31 2.5 Genomic DNA extraction ............................................................................................... 31 2.6 Southern blots for telomere addition and length ............................................................ 31 iv 2.7 PCR mutagenesis screens ............................................................................................... 32 2.8 Chromatin mobility analysis .......................................................................................... 33 2.9 Antibodies and immunoblotting ..................................................................................... 33 2.10 Cell cycle arrest .............................................................................................................. 34 2.11 Recombinant protein production .................................................................................... 34 2.12 Rad53 kinase reactions ................................................................................................... 35 2.13 Visualization of kinetochores ......................................................................................... 35 2.14 Analysis of SPB-CEN dynamics .................................................................................... 36 2.15 DNA damage sensitivity ................................................................................................ 36 2.16 HR repair analysis .......................................................................................................... 36 2.17 Checkpoint analysis ........................................................................................................ 37 2.18 A-like faker (ALF) assay ................................................................................................ 37 2.19 Chromosome transmission fidelity (CTF) assay ............................................................ 37 2.20 Chromatin immunoprecipitation (ChIP) ........................................................................ 38 2.21 Peptide pulldown assays ................................................................................................. 38 2.22 Break-induced replication (BIR) assay .......................................................................... 38 2.23 Break-induced replication PCR assay ............................................................................ 39 2.24 Cell senescence assays ................................................................................................... 39 2.25 Yeast two-hybrid ............................................................................................................ 39 2.26 Statistics ......................................................................................................................... 40 Chapter 3 A Pif1-dependent threshold separates DSBs and telomeres ........................................ 41 3.1 Introduction ........................................................................................................................ 42 3.2 Results ................................................................................................................................ 42 3.2.1 Identification of a threshold for Pif1 sensitivity ......................................................... 42 3.2.2 Pif1 is not inhibited by DNA damage kinases ...........................................................
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