HECTD1 promotes base excision repair in nucleosomes through chromatin remodelling

Bennett, Laura, Madders, Eleanor CET and Parsons, Jason L ORCID: 0000-0002-5052-1125 (2020) HECTD1 promotes base excision repair in nucleosomes through chromatin remodelling. Nucleic Acids Research, 48 (03). pp. 1301-1313.

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Official URL: https://academic.oup.com/nar/article/48/3/1301/565...

Abstract

Base excision repair (BER) is the major cellular DNA repair pathway that recognises and excises damaged DNA bases to help maintain genome stability. Whilst the major enzymes and mechanisms co-ordinating BER are well known, the process of BER in chromatin where DNA is compacted with histones, remains unclear. Using reconstituted mononucleosomes containing a site-specific synthetic abasic site (tetrahydrofuran, THF), we demonstrate that the DNA damage is less efficiently incised by recombinant AP endonuclease 1 (APE1) when the DNA backbone is facing the histone core (THF-in) compared to that orientated away (THF-out). However, when utilizing HeLa whole cell extracts, the difference in incision of THF-in versus THF-out is less pronounced suggesting the presence of chromatin remodelling factors that stimulate THF accessibility to APE1. We subsequently purified an activity from HeLa cell extracts and identify this as the E3 ubiquitin ligase, HECTD1. We demonstrate that a recombinant truncated form of HECTD1 can stimulate incision of THF-in by APE1 in vitro by histone ubiquitylation, and that siRNA-mediated depletion of HECTD1 leads to deficiencies in DNA damage repair and decreased cell survival following x-ray irradiation, particularly in normal fibroblasts. Thus, we have now identified HECTD1 as an important factor in promoting BER in chromatin.

Item Type:	Article
Uncontrolled Keywords:	Hela Cells, Chromatin, Nucleosomes, Humans, DNA Damage, Furans, DNA-(Apurinic or Apyrimidinic Site) Lyase, Ubiquitin-Protein Ligases, DNA Polymerase beta, Histones, DNA, Chromatin Assembly and Disassembly, DNA Repair
Depositing User:	Symplectic Admin
Date Deposited:	05 Dec 2019 09:34
Last Modified:	19 Jan 2023 00:16
DOI:	10.1093/nar/gkz1129
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3064830