Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses

DNA Repair (Amst). 2013 Sep;12(9):774-85. doi: 10.1016/j.dnarep.2013.06.004. Epub 2013 Jul 18.


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.

Keywords: DNA polymerase β; Methyl methanesulfonate; PARP inhibitor; PARP-1; Poly(ADP-ribose); XRCC1.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 1-Naphthylamine / analogs & derivatives
  • 1-Naphthylamine / pharmacology
  • Animals
  • Antineoplastic Agents, Alkylating / pharmacology
  • Cell Cycle / drug effects
  • Cells, Cultured
  • DNA Damage*
  • DNA Polymerase beta / metabolism
  • DNA Repair
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Hydrogen Peroxide / pharmacology
  • Inhibitory Concentration 50
  • Methyl Methanesulfonate / pharmacology
  • Mice
  • Mice, Knockout
  • Models, Molecular
  • Mutation, Missense
  • Naphthalimides / pharmacology
  • Nuclear Magnetic Resonance, Biomolecular
  • Oxidants / pharmacology
  • Oxidation-Reduction
  • Poly (ADP-Ribose) Polymerase-1
  • Poly Adenosine Diphosphate Ribose / metabolism
  • Poly(ADP-ribose) Polymerase Inhibitors
  • Poly(ADP-ribose) Polymerases / metabolism*
  • Protein Binding
  • Protein Structure, Tertiary
  • Quinolones / pharmacology
  • Transition Temperature
  • X-ray Repair Cross Complementing Protein 1


  • Antineoplastic Agents, Alkylating
  • DNA-Binding Proteins
  • Naphthalimides
  • Oxidants
  • Poly(ADP-ribose) Polymerase Inhibitors
  • Quinolones
  • X-ray Repair Cross Complementing Protein 1
  • Xrcc1 protein, mouse
  • 4-amino-1,8-naphthalimide
  • Poly Adenosine Diphosphate Ribose
  • 1-Naphthylamine
  • Methyl Methanesulfonate
  • Hydrogen Peroxide
  • Parp1 protein, mouse
  • Poly (ADP-Ribose) Polymerase-1
  • Poly(ADP-ribose) Polymerases
  • DNA Polymerase beta