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The Effect of Telomeric Repeats on Double-Strand Break Processing and Repair

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The Effect of Telomeric Repeats on Double-Strand Break Processing and Repair The effect of telomeric repeats on double-strand break processing and repair Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Isabella Marcomini aus Italien Basel, 2018 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Susan M. Gasser Prof. Dr. Joachim Lingner Basel, den 27. März 2018 Prof. Dr. Martin Spiess Dekan 2 Table of Contents Thesis overview: ..................................................................................................................................... 5 Chapter 1: An Introduction to DNA end-processing: telomeres versus double- strand breaks............................................................................................................................................ 7 The double strand break response............................................................................................ 9 The nature of telomeres .............................................................................................................. 11 Double strand break repair proteins with telomeric functions ............................... 12 Regulation of telomerase ............................................................................................................ 14 Control of DNA end resection .................................................................................................. 16 The processing of TG-flanked DSBs ....................................................................................... 19 Persistent DSBs and telomeres share common nuclear subcompartments ...... 20 Nuclear organization of telomeres .............................................................................. 20 Nuclear organization in DSB processing..................................................................23 Aim of the thesis...........................................................................................................................26 References............................................................................................................................................27 Chapter 2: Processing of DSBs at the nuclear periphery ................................................ 35 SWR1 and INO80 chromatin remodelers contribute to DNA double-strand break perinuclear anchorage site choice................................................................................35 PolySUMOylation by Siz2 and Mms21 triggers relocation of DNA breaks to nuclear pores through the Slx5/Slx8 STUbL...............................................................65 Chapter 3: Telomeric repeats near an internal double-strand break prevent MRX recruitment and allow DNA end separation to favor ectopic recombination ...... 101 Chapter 4: Conclusions and future perspectives..............................................................133 Processing of a persistent DSB...........................................................................................134 A different approach to investigate repair at the nuclear periphery........ 134 The nucleo- to cytoskeleton connection.................................................................135 Processing of TG-flanked DSBs...........................................................................................137 The resection block imposed by TG repeats......................................................... 138 Control of chromosome length by SMC proteins................................................139 A possible role of transcription in nuclear organization of DNA damage.................................................................................................................................142 The role of end separation in repair pathway choice......................................143 Relevant modifications to the TG-HO system......................................................145 A model system for the study of translocations................................................. 147 References........................................................................................................................................ 148 Appendices ........................................................................................................................................... 153 List of abbreviations .................................................................................................................... 153 Curriculum vitae ........................................................................................................................... 155 Acknowledgments ....................................................................................................................... 159 3 4 Thesis overview This PhD thesis is based on the following publications: • Horigome C, et al. (2014). SWR1 and INO80 chromatin remodelers contribute to DNA double-strand break perinuclear anchorage site choice. Molecular Cell 55(4): 626-39 • Horigome C, et al. (2016). PolySUMOylation by Siz2 and Mms21 triggers relocation of DNA breaks to nuclear pores through the Slx5/Slx8 STUbL. Genes & Development 30(8): 931-45 • Marcomini I, Gasser SM (2015). Nuclear organization in DNA end processing: Telomeres vs double-strand breaks. DNA Repair 32: 134-40 • Marcomini I, et al. (2018). Asymmetric processing of DNA ends at a double- strand break leads to unconstrained dynamics and ectopic translocation. Cell reports 24: 2614-28 This thesis consists of 4 chapters. Chapter 1 is an introduction, based on a published review (Marcomini I, Gasser SM, 2015). Chapter 2 presents the published papers on DSB processing relative to the nuclear periphery in S. cerevisiae, to which I contributed. Chapter 3 is the published article about the effect on telomeric repeats on DSB processing. Chapter 1 gives an overview of the field of end processing in the double-strand break response as well as at telomeres. It is based on a review (Marcomini and Gasser, 2015), but is rearranged, updated and modified in order to avoid redundancy and provide clarity. Additional relevant sections and citations were added that were not adequately discussed in the review or outdated since its publication. Chapter 2 is an experimental chapter that reports previous works on the role of the nuclear periphery in DSB processing. These studies provided a foundation to develop my own project, and they are some of the first works showing a role for the nuclear periphery in the DNA damage response. While the first one (2014) dissects the requirements for a persistent DSB to interact with the nuclear envelope, the second one focuses on the role and extent of SUMOylation in such interactions, providing functional data for their biological relevance. I participated in both papers (Horigome C et al., 2014 and 2016), which were lead by Dr Horigome. Chapter 3 presents the paper as published on Cell reports. This work stems from the basic question of what determines the identity of a DNA end as a telomere. It isolates telomeric repeats in an internal chromosomal position next to a DSB end, and analyzes their effects on the processing, nuclear localization and repair of the DSB. Finally, Chapter 4 discusses the significance of these studies in the context of DNA repair and telomere biology, highlights open questions and provides future perspectives. 5 6 CHAPTER 1. AN INTRODUCTION TO DNA END PROCESSING: TELOMERES VERSUS DOUBLE- STRAND BREAKS Based on: Marcomini I and Gasser S.M. DNA Repair 32:134-40, 2015 SUMMARY DNA double strand breaks (DSBs) can arise in eukaryotic cells due to exogenous (radiation, chemicals) or endogenous (oxidative stress, replication fork collapse) processes. DSBs consitute a threat for genome integrity and need to be repaired to avoid loss of genetic material and allow cell cycle progression. Telomeres are the ends of eukaryotic chromosome and, like DSBs, expose a free 3’-OH. What differentiates telomeres from DSBs has been a long-standing question in the field of cell biology: indeed, recognition of telomeres as DSBs would cause chromosome fusions and genomic instability. Unlike DSBs, telomeres are protected from the DNA damage response machinery by a dedicated protein complex, the telosome, as well as by their association in clusters at the nuclear periphery. Interestingly, some components of the telosome have fundamental but different functions in DSB processing. Furthermore, some nuclear envelope binding sites are common to telomeres and the processing of DSBs. This introduction highlights common pathways as well as specific features of these two different kinds of DNA ends. 7 8 Introduction The double-strand break response DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage in the nucleus, particularly if an intact template is not immediately available for repair(Jackson and Bartek 2009). To ensure cell survival, DSBs trigger a conserved cascade of events called the DNA damage checkpoint, that arrests the cell cycle and stimulates repair(Tsabar and Haber 2013). The Ser/Thr PI3-like kinases ATM (Tel1 in the budding yeast, S. cerevisiae) and ATR (Mec1 in S. cerevisiae) initiate the checkpoint signaling by phosphorylating other effector kinases, thereby promoting the recruitment of the repair machinery, the stalling of cell cycle progression, and changes in gene expression(Finn et al. 2011; Smeenk
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