HECTD1 promotes base excision repair in nucleosomes through chromatin remodelling
Allbwn ymchwil: Cyfraniad at gyfnodolyn › Erthygl › adolygiad gan gymheiriaid
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Yn: Nucleic Acids Research, Cyfrol 48, Rhif 3, 20.02.2020, t. 1301-1313.
Allbwn ymchwil: Cyfraniad at gyfnodolyn › Erthygl › adolygiad gan gymheiriaid
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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 -