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Title: Epigenetic mechanisms involved in the cellular response to DNA damage processed by Base Excision Repair
Author: Bennett, L.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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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 contain 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 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.
Supervisor: Parsons, J. L. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral