Abstract
XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POLβ and PNK colocalize with XRCC1 in replication foci and that POLβ and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLβ and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLβ to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc.
Highlights
X-ray repair cross-complementing protein 1 (XRCC1) has no known enzymatic activity but is involved in single-strand break repair (SSBR) and base excision repair (BER), both considered to be part of the same pathway
Neutralising antibodies against polymerase b (POLb) strongly inhibited the total BER capacity of the XRCC1-EYFP immunoprecipitates after 15 min of repair (Supporting Information Figure S1C). These results suggest that in the XRCC1-EYFP complexes, POLb is more important for the initial single nucleotide BER reaction, while POLd is more important after prolonged incubation and for LP repair synthesis
Our results (Table I and Fig. 3C) indicate that the level of XRCC1 in the foci affects the recruitment and/or the retention of poly (ADP-ribose) polymerase 1 (PARP-1) in micro-irradiated regions. To explore whether this was due to the level of polyADPribosylation (PARylation), we examined the effect of PARP-inhibitors on the recruitment of DNA repair proteins to the site of DNA damage
Summary
X-ray repair cross-complementing protein 1 (XRCC1) has no known enzymatic activity but is involved in single-strand break repair (SSBR) and base excision repair (BER), both considered to be part of the same pathway. Base damage of different types, abasic sites (AP sites), and SSBs are continuously formed in the human genome in numbers exceeding 104 per day. Repair of such lesions involves a number of proteins in addition to XRCC1 and is mainly carried out by the BER/SSBR pathway.
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