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 ? (pol ?) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol ? in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol ? is found in vivo, and it is known that pol ? 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 ?, 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 ? (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 ? in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.

SUBMITTER: Horton JK 

PROVIDER: S-EPMC3924596 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature