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«Exploring DNA Damage Induced Foci and Their Role in Coordinating the DNA Damage Response By ManTek Yeung A thesis submitted in conformity with the ...»

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Exploring DNA Damage Induced Foci and Their Role in

Coordinating the DNA Damage Response

By

ManTek Yeung

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Department of Molecular Genetics

University of Toronto

© Copyright by ManTek Yeung 2012

Exploring DNA Damage Induced Foci and Their Role in

Coordinating the DNA Damage Response

ManTek Yeung

Doctor of Philosophy

Department of Molecular Genetics

University of Toronto

Abstract

DNA damage represents a major challenge to the faithful replication and transmission of genetic information from one generation to the next. Cells utilize a highly integrated network of pathways to detect and accurately repair DNA damage. Mutations arise when DNA damage persists undetected, unrepaired, or repaired improperly. Mutations are a driving force of carcinogenesis and therefore many of the DNA damage surveillance and repair mechanisms guard against the transformation of normal cells into cancer cells.

Central to the detection and repair of DNA damage is the relocalization of DNA damage surveillance proteins to DNA damage where they assemble into subnuclear foci and are capable to producing a signal that the cell interprets to induce cellular modifications such as cycle arrest and DNA repair which are important DNA damage tolerance. In this work, I describe my quest to understand the mechanisms underlying the assembly, maintenance, and disassembly of these DNA damage-induced foci and how they affect DNA damage signaling in Saccharomyces cerevisiae. First, I describe phenotypic characterization of a novel mutation that impairs assembly of the 9-1-1 checkpoint clamp complex into foci.

ii Second, I describe my work to further understand the roles of the histone phosphatase Pph3 and phosphorylated histone H2A in modulating DNA damage signaling. Third, I include my work to uncover the possible mechanism by which the helicase Srs2 works to enable termination of DNA damage signaling. In summary, this thesis documents my efforts to understand the cellular and molecular nature of DNA damage signaling and how signaling is turned off in coordination with DNA damage repair.

iii Acknowledgments I would like to thank my supervisor Dr. Daniel Durocher for his guidance and mentorship throughout my time in the lab. Dan, your passion for science has always been an inspiration for me. I would also like to thank my committee members, Dr. Charlie Boone and Dr. Brigitte Lavoie, for their guidance throughout my graduate training. Thank you to my friends who stopped asking me when I was going to graduate in 2009. And finally, I would like to thank my parents who have been unconditionally loving and supportive all these years in my endeavors.

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2.3 Rad17-K197 Ddc1-GFP focus formation and localization to damaged 67 chromatin rad17-K197R and rad18∆ moderately impair checkpoint signaling at DSBs 80 2.4

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Reversal of post-translational modification to enable checkpoint recovery 193 Synthetic genetic screens and proteomic approaches to understand γH2A 200 function Identifying novel dpb11 alleles that suppress srs2∆ 202

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Figure 1.3.

1 The DNA damage checkpoint at a double-strand break in S. cerevisiae 8 Figure 1.3.

2 Rad53 and Chk1 cooperatively inhibit cell cycle progression 17 Figure 1.4.

1 Homologous recombination pathways for double-strand break repair 28

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Figure 3.5.

1 Tetrad analysis of pph3::NAT hta1-S129A hta2-S129A x mre11::KAN. 129 Figure 3.5.

2 Tetrad analysis of pph3::NAT x dia2::KAN and pph3::NAT hta1-S129A 130

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Figure 3.6.

1 h2a-S129A elevates levels of spontaneous Ddc2-GFP and Rad52-YFP foci 132 Figure 3.6.

2 Fluctuation analysis of gross chromosome rearrangement frequency 134 Figure 4.2.

1 Ddc2-GFP focus dynamics following DSB induction and repair 153 Figure 4.2.

2 onitoring recovery from the DNA damage checkpoint as measured by 155

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Figure 4.2.

3 Ddc1-RFP focus dynamics following DSB induction and repair 157 Figure 4.2.

4 Dpb11-GFP focus dynamics following DSB induction and repair 159 Figure 4.3.1 Chromatin association of Ddc2-13myc following DSB induction and repair 162 Figure 4.3.2 Chromatin association of Ddc1-13myc following DSB induction and repair 165 Figure 4.4.





1 Rfa1-CFP and Ddc2-YFP foci dynamics following the induction and repair 166

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Figure 4.5.

1 Persistent chromatin association of Rad51 in srs2∆ strains 179 Figure 4.5.

2 Survival of YMV2 pol32∆ and srs2∆ cells on galactose media 184

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Appendix 2 Ddc2-GFP foci during recovery in asf1 cells 234 Appendix 3 An RFA-3HA tag rescues srs2Δ lethality to a DSB 236 Appendix 4 Ddc1-GFP focus accumulation after CDK inhibition 238

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Introduction

1.1 DNA damage, Genome stability, and Cancer Faithful transmission of genetic information from one generation to the next requires the efficient recognition and accurate repair of DNA damage. Incomplete or inaccurate repair of DNA damage results in mutations or cell death. An extensive cellular network called the DNA damage response (DDR) reacts to DNA damage and coordinates cell cycle arrest, global changes in transcription, chromatin remodeling, DNA repair, and—in metazoans—apoptosis (Jackson and Bartek, 2009). Central to the DDR is an evolutionarily conserved signal transduction cascade called the DNA damage checkpoint.

The signaling cascade prevents cell cycle progression until DNA damage is repaired.

Mutations in the genes that regulate the checkpoint and the DDR can result in genome instability, a hallmark of cancer (Kolodner et al., 2002; Harper and Elledge, 2007).

Therefore, understanding the genetic and molecular mechanisms that control the DNA damage response is important for understanding how cancer develops and how cancer cells thrive.

The DNA damage checkpoint is well-conserved across all organisms. While most of the initial understanding of the mechanisms responsible for detection, signaling, and repair of DNA damage originated from studies in single-celled organisms such as bacteria, Schizosaccharomyces pombe, and Saccharomyces cerevisiae, research efforts in the last 10-15 years have yielded great progress in elucidating the DNA damage response in complex metazoans including humans.

A conserved phenomenon of the DNA damage response seen from yeast to mammals is the reorganization of checkpoint and repair proteins into punctate subnuclear foci that are detectable by fluorescence microscopy (Lisby and Rothstein, 2005). Foci were first observed with DNA repair proteins in budding yeast and mammalian cells (Bishop, 1994; Haaf et al., 1995). DNA damage foci are important for the proper execution of the DDR, as they allow a rapid and local increase in protein concentration around DNA damage without the need for energetically costly and mechanistically slow up-regulation of gene expression. Furthermore, foci have been widely used as markers of DNA damage and the discovery of the genetic requirements for foci assembly have greatly advanced our understanding of the DDR (Melo et al., 2001; Lisby et al., 2004;

Kolas et al., 2007). In contrast, how foci are subsequently disassembled was virtually unexplored when I began in the lab. Indeed, it was unknown if focus disassembly was even a requirement for termination of checkpoint signaling. In this thesis, I will describe my efforts to understand the molecular mechanisms underlying foci assembly and disassembly in coordination with DNA damage checkpoint signaling. This chapter will explore the highly conserved DNA damage response in Saccharomyces cerevisiae with reference to the homologs in humans and other species.

1.2 Types of DNA damage DNA molecules are constantly subjected to lesions that can impede replication, transcription, or chromosome segregation. DNA can be damaged by exogenous agents such as ultraviolet (UV) radiation, ionizing radiation (IR), X-rays, and DNA-damaging chemicals. Endogenous sources of DNA damage arise from normal cellular metabolism which generates reactive oxygen species that react with the DNA molecule.

Types of DNA damage include single base pair modifications, single-strand breaks, interstrand crosslinks, and double-strand breaks (DSBs). Many different agents are used in the laboratory to study the cellular response to different types of DNA damage. Covalent modifications to nucleotides that result from methyl methanesulfonoate (MMS), mitomycin C, or UV radiation will impede DNA replication and transcription (Cadet et al., 1997; Lundin et al., 2005). The chemical bleomycin and IR are routinely used to generate DSBs. DSBs are the most toxic DNA lesions, as a single DSB is lethal to a cell (Sandell and Zakian, 1993). Left unrepaired or repaired improperly, the result can be aneuploidy, loss of the acentric portion of the chromosome and gross chromosome rearrangements including translocations and inversions (Kolodner et al., 2002). More generally, induction of DNA damage with DNA damaging compounds such as ethyl methane sulfonoate (EMS) are used for mutagenesis in genetic screens that address a diverse set of biological questions. Much of the work presented in this thesis focuses on the cellular response to DSBs, the most toxic of DNA lesions (Sandell and Zakian, 1993).

DNA replication creates major challenges to genome stability (Branzei and Foiani, 2010). Imperfect replication by DNA polymerases can result in base-pair mismatches or strand slippage in highly repetitive regions of the genome, changing the number of repeats at a locus. Changes in sequence copy number are associated with severe disorders including Huntington’s disease (Price et al., 1998). DNA damage creates barriers to replication and when a replisome encounters DNA damage, it can collapse into a DSB (Branzei and Foiani, 2005). DNA damage to nucleotides, ssDNA breaks, and interstrand crosslinks are barriers to replisome progression. In addition, DNA-protein complexes at highly transcribed genes, centromeres, and telomeres create natural barriers to replication (Ivessa et al., 2003). These domains are prone to replication fork collapse and DSBs (Szilard et al., 2010). Hydroxyurea (HU) is a chemical frequently used to induce replication stress by limiting the availability of dNTPs for DNA synthesis. Within S-phase, there is a replication checkpoint (or S-phase checkpoint) that shares many common components with the DNA damage checkpoint and stabilizes the replication fork to prevent fork collapse into a DSB (Lopes et al., 2001).

While DNA damage poses a major risk to the faithful transmission of genetic information, cells utilize controlled DNA damage to accomplish a variety of biological functions. During meiosis, DSBs are created to initiate homologous recombination between homologous chromosomes. In budding yeast, a DSB is induced in the MAT locus by the homothallic (HO) endonuclease which is repaired by homologous donor sequences HMRa or HMLα to accomplish mating type switching (Haber, 1998b). In vertebrates, the generation of antibodies requires the induction of a DSB in the V(D)J locus to promote class-switch recombination, a critical process that generates diverse antigen recognition by the immune system (Dudley et al., 2005). DDR pathways and programmed DSB pathways are intimately related. Often, mutations in DDR genes have pathological consequences in programmed DSB pathways as well, including immunodeficiency or sterility in animals.

1.3 The DNA damage checkpoint The DNA damage checkpoint is a signaling transduction cascade The DNA damage checkpoint is activated in the presence of DNA damage and arrests the cell cycle (Zhou and Elledge, 2000). In yeast, a single DSB is sufficient to activate the DNA damage checkpoint and arrest the cell in metaphase just prior to chromosome segregation (Sandell and Zakian, 1993). By convention, this arrest is called “G2/M arrest” (Sandell and Zakian, 1993). The first DNA damage checkpoint gene identified in budding yeast was RAD9 and was found to be essential for cell cycle arrest at G2/M following DNA damage (Weinert and Hartwell, 1988). Later discoveries revealed that the DNA damage checkpoint can control cell cycle progression in other phases of the cell cycle (Painter and Young, 1980) and many components of the checkpoint work to stabilize a replication fork in S-phase (Paulovich and Hartwell, 1995).

DNA damage checkpoint proteins can be classified by their specific functions within the signaling cascade: sensors, transducers, mediators, and effectors (Table 1.3.1) (Melo and Toczyski, 2002). Many of these key players are conserved through evolution, although many important differences between organisms have also been discovered. The checkpoint is a tightly choreographed response that coordinates many cellular changes.



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