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Epigenetic Mechanisms Involved in the Cellular Response to DNA Damage Processed by Base Excision Repair

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Epigenetic mechanisms involved in the cellular response to DNA damage processed by Base Excision Repair Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Laura Gail Bennett November 2017 i Abstract Chromatin remodelling is required for access to occluded sequences of DNA by proteins involved in important biological processes, including DNA replication and transcription. There is an increasing amount of evidence for chromatin remodelling during DNA repair, although this has been mostly focused towards DNA double strand break and nucleotide excision repair. At this time there is little evidence for chromatin remodelling in base excision repair (BER). BER is a highly conserved DNA repair pathway which processes spontaneous endogenous DNA base damages generated by oxidative metabolism, but also those induced by exogenous agents (eg. ionising radiation), to maintain genome stability. The mechanism in which the BER repairs damaged bases has been extensively studied and the repair proteins involved are well known. However in terms of chromatin, BER is poorly understood. It is thought that chromatin remodelling occurs due to accumulating evidence indicating that certain BER enzymes are significantly less efficient at acting on sterically occluded sites and near the nucleosome dyad axis. At this time the mechanisms and enzymes involved to facilitate BER are unknown. Therefore, the study presented in this thesis aimed to identify specific histone modification enzymes and/or chromatin remodellers that are involved in the processing of DNA base damage during BER. A method to generate two mononucleosome substrates with a site specific synthetic AP site (tetrahydrofuran; THF) was used to measure recombinant AP endonuclease 1 (APE1) activity alone, and APE1 in HeLa whole cell extract (WCE) that contained chromatin modifiers. The substrates contained either a THF rotationally positioned in the mononucleosome so the DNA backbone was facing outwards (THF-OUT) so accessible to APE1, or facing inwards (THF- IN) towards the histone octamer and so sterically occluded to APE1. I discovered that the THF-OUT substrate was efficiently processed by recombinant APE1 alone and by APE1 in HeLa WCE. In contrast, recombinant APE1 activity was significantly impeded by THF-IN, but which was efficiently processed by APE1 in HeLa WCE in the presence of factors supporting ubiquitination. This suggested the presence of a chromatin modifier, predictably E3 ubiquitin ligase(s) present in WCE that was increasing THF-IN accessibility to APE1. A sequential chromatography approach was utilised to purify these novel activities from HeLa WCE, and I identified three separate activities capable of stimulating APE1 activity towards the THF-IN mononucleosome. Y-box protein 3 (YBX3) and HECT Domain E3 Ubiquitin Protein Ligase 1 (HECTD1) were identified by mass spectrometry analysis of active fractions and their presence aligned with the APE1 stimulatory activity profile of the THF-IN substrate. Depletion of these proteins using siRNA in HeLa cells decreased cell survival following ionising radiation, and delayed DNA damage repair in both HeLa cells and in normal lung fibroblasts. Together ii these results suggest that HECTD1 and YBX3 are strong candidates required to facilitate BER through histone ubiquitination and/or chromatin remodelling, and provide new mechanistic information on the process of BER in cellular chromatin. iii Acknowledgements This PhD thesis becomes reality with the kind support and help of many individuals. I would like to gratefully acknowledge various people who have been instrumental in the completion of this thesis. I would like to acknowledge the University of Liverpool for funding this project. First and foremost, I would like to thank my supervisor, Dr Jason Parson for his encouragement, expertise, motivation and patience during this project, and for instilling confidence in myself and my work. I am very grateful for the valuable comments and proofreading of this thesis. I have been very lucky to have been part of a research team with helpful and supportive members who have helped me survive and kept me (moderately) sane. I would particularly like to thank Dr Katie Nickson, Dr Matthew Edmonds, Dr Rachel Carter and Sarah Williams for their advice and encouragement. I am also grateful to all other members of the group and other teams in the North West Cancer Research Centre. Finally, I must express my very profound gratitude to my long-suffering parents, my sister, pets and friends for providing me with unfailing support and continuous encouragement throughout my years of study, research and writing of this thesis. This accomplishment would not have been possible without them. iv Table of Contents Abstract ..................................................................................................................................... i Acknowledgements ..................................................................................................................iii Table of Contents .................................................................................................................... iv List of Tables ......................................................................................................................... xvii Abbreviations ....................................................................................................................... xviii CHAPTER I .............................................................................................................................. 1 INTRODUCTION ................................................................................................................. 1 1.1 Genome Stability and Instability ................................................................................. 1 1.1.1 Deoxyribose Nucleic acid .................................................................................. 2 1.1.2 DNA Damage ..................................................................................................... 5 1.1.3 Endogenous DNA damage ............................................................................... 6 1.1.3.1 Hydrolysis ................................................................................................... 6 1.1.3.2 Oxidation ..................................................................................................... 7 1.1.3.3 Errors from DNA Processing ...................................................................... 8 1.1.4 Exogenous damage ........................................................................................... 8 1.1.4.1 UV ............................................................................................................... 8 1.1.4.2 Ionising Radiation ....................................................................................... 9 1.1.4.3 Alkylation .................................................................................................... 9 1.1.5 Common types of DNA damage .................................................................. 10 1.1.5.1 Double strand breaks................................................................................ 10 1.1.5.2 Single strand breaks ................................................................................. 11 1.1.5.3 AP sites ..................................................................................................... 12 1.1.5.4 8-oxoG ...................................................................................................... 13 1.1.5.5 Thymine Glycol ......................................................................................... 14 1.2 DNA Repair ............................................................................................................. 15 1.2.1 Double strand break repair ............................................................................ 15 v 1.2.1.1 Homologous Recombination .................................................................... 16 1.2.1.2 Non-homologous end-joining .................................................................... 17 1.2.2 Nucleotide excision repair ............................................................................... 19 1.2.2.1 GG-NER Recognition ............................................................................... 20 1.2.2.2 TC-NER Recognition ................................................................................ 20 1.2.2.3 Incision, excision, gap filling and ligation .................................................. 21 1.2.3 Base excision repair ......................................................................................... 22 1.2.3.1 Base Removal .......................................................................................... 23 1.2.3.2 AP Site Incision ......................................................................................... 25 1.2.3.3 PARP1 and single strand break repair ..................................................... 26 1.2.3.4 End Processing ......................................................................................... 27 1.2.3.5 Gap Filling ................................................................................................. 28 1.2.3.6 Nick Sealing .............................................................................................. 29 1.2.3.7 Base excision repair and Cancer .............................................................
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