title: Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses.
abstract: Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12-C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1(-/-) mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.
authors.affiliation: Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 29909-2233, USA.
authors.forename: Julie K
authors.initials: JK
authors.lastname: Horton
authors.forename: Donna F
authors.initials: DF
authors.lastname: Stefanick
authors.forename: Natalie R
authors.initials: NR
authors.lastname: Gassman
authors.forename: Jason G
authors.initials: JG
authors.lastname: Williams
authors.forename: Scott A
authors.initials: SA
authors.lastname: Gabel
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cuneo
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
authors.forename: Padmini S
authors.initials: PS
authors.lastname: Kedar
authors.forename: Eugene F
authors.initials: EF
authors.lastname: Derose
authors.forename: Esther W
authors.initials: EW
authors.lastname: Hou
authors.forename: Robert E
authors.initials: RE
authors.lastname: London
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D015057:1-Naphthylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D020125:Mutation, Missense; D053644:Naphthalimides / Q000494:pharmacology; D019906:Nuclear Magnetic Resonance, Biomolecular; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D000071137:Poly (ADP-Ribose) Polymerase-1; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015363:Quinolones / Q000494:pharmacology; D044366:Transition Temperature; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D018906:Antineoplastic Agents, Alkylating; D004268:DNA-Binding Proteins; D053644:Naphthalimides; D016877:Oxidants; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D015363:Quinolones; D000076105:X-ray Repair Cross Complementing Protein 1; C000619352:Xrcc1 protein, mouse; C086538:4-amino-1,8-naphthalimide; D011064:Poly Adenosine Diphosphate Ribose; D015057:1-Naphthylamine; D008741:Methyl Methanesulfonate; D006861:Hydrogen Peroxide; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D019951:DNA Polymerase beta
keywords: DNA polymerase β; Methyl methanesulfonate; PARP inhibitor; PARP-1; Poly(ADP-ribose); XRCC1