TY - JOUR

T1 - HECTD1 promotes base excision repair in nucleosomes through chromatin remodelling

AU - Bennett, Laura

AU - Madders, Eleanor C E T

AU - Parsons, Jason L

N1 - © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

PY - 2020/2/20

Y1 - 2020/2/20

N2 - 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.

AB - 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.

KW - Chromatin/genetics

KW - Chromatin Assembly and Disassembly/genetics

KW - DNA/genetics

KW - DNA Damage/drug effects

KW - DNA Polymerase beta/genetics

KW - DNA Repair/genetics

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics

KW - Furans/pharmacology

KW - HeLa Cells

KW - Histones/genetics

KW - Humans

KW - Nucleosomes/genetics

KW - Ubiquitin-Protein Ligases/genetics

U2 - 10.1093/nar/gkz1129

DO - 10.1093/nar/gkz1129

M3 - Article

C2 - 31799632

VL - 48

SP - 1301

EP - 1313

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 3

ER -