DNA damage levels and biochemical repair capacities associated with XRCC1 deficiency.
Base excision repair (BER) is the major corrective pathway for most spontaneous, oxidative, and alkylation DNA base and sugar damage. X-ray cross-complementing 1 (XRCC1) has been suggested to function at nearly every step of this repair process, primarily through direct protein-protein interactions. Using whole cell extract (WCE) repair assays and DNA damage measurement techniques, we examined systematically the quantitative contribution of XRCC1 to specific biochemical steps of BER and single-strand break repair (SSBR). Our studies reveal that XRCC1-deficient Chinese hamster ovary WCEs exhibit normal base excision activity for 8-oxoguanine (8-OH-dG), 5-hydroxycytosine, ethenoadenine, and uracil lesions. Moreover, XRCC1 mutant EM9 cells possess steady-state levels of endogenous 8-OH-dG base damage similar to those of their wild-type counterparts. Abasic site incision activity was found to be normal in XRCC1-deficient cell extracts, as were the levels of abasic sites in isolated chromosomal DNA from mutant cells. While one- and five-nucleotide gap filling was not affected by XRCC1 status, a significant approximately 2-4-fold reduction in nick ligation activity was observed in EM9 WCEs. Our results herein suggest that the primary biochemical defect associated with XRCC1 deficiency is in the ligation step of BER/SSBR, and that XRCC1 plays no significant role in endogenous base damage and abasic site repair, or in promoting the polymerase gap-filling step.
Wong
Heng-Kuan
HK
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
Kim
Daemyung
D
Hogue
Barbara A
BA
McNeill
Daniel R
DR
Wilson
David M
DM
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D006224:Cricetinae; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000172:deficiency* / Q000378:metabolism; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D009154:Mutation; D010053:Ovary / Q000166:cytology; D013997:Time Factors; D014498:Uracil / Q000031:analogs & derivatives / Q000378:metabolism; D000076105:X-ray Repair Cross Complementing Protein 1
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C024829:8-hydroxyguanine; D014498:Uracil; D006147:Guanine; C017400:5-hydroxycytosine; D003596:Cytosine; D000225:Adenine