title: Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin.
abstract: A critical step of DNA single-strand break repair is the rapid recruitment of the scaffold protein XRCC1 that interacts with, stabilizes and stimulates multiple enzymatic components of the repair process. XRCC1 recruitment is promoted by PARP1, an enzyme that is activated following DNA damage and synthesizes ADP-ribose polymers that XRCC1 binds directly. However, cells possess two other DNA strand break-induced PARP enzymes, PARP2 and PARP3, for which the roles are unclear. To address their involvement in the recruitment of endogenous XRCC1 into oxidized chromatin we have established 'isogenic' human diploid cells in which PARP1 and/or PARP2, or PARP3 are deleted. Surprisingly, we show that either PARP1 or PARP2 are sufficient for near-normal XRCC1 recruitment at oxidative single-strand breaks (SSBs) as indicated by the requirement for loss of both proteins to greatly reduce or ablate XRCC1 chromatin binding following H2O2 treatment. Similar results were observed for PNKP; an XRCC1 protein partner important for repair of oxidative SSBs. Notably, concentrations of PARP inhibitor >1000-fold higher than the IC50 were required to ablate both ADP-ribosylation and XRCC1 chromatin binding following H2O2 treatment. These results demonstrate that very low levels of ADP-ribosylation, synthesized by either PARP1 or PARP2, are sufficient for XRCC1 recruitment following oxidative stress.
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9RQ, UK.
authors.forename: Hana
authors.initials: H
authors.lastname: Hanzlikova
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9RQ, UK.
authors.forename: William
authors.initials: W
authors.lastname: Gittens
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9RQ, UK.
authors.forename: Katerina
authors.initials: K
authors.lastname: Krejcikova
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9RQ, UK.
authors.forename: Keith W
authors.initials: KW
authors.lastname: Caldecott
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism*; D053904:DNA Breaks, Single-Stranded; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D051379:Mice; D018384:Oxidative Stress; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D000071137:Poly (ADP-Ribose) Polymerase-1 / Q000235:genetics / Q000502:physiology*; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000502:physiology*; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; C000619352:Xrcc1 protein, mouse; C499320:PARP1 protein, human; C483041:PARP2 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C530380:PNKP protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); D045643:DNA Repair Enzymes