The Arg280His polymorphism in X-ray repair cross-complementing gene 1 impairs DNA repair ability

The contribution of three single nucleotide polymorphisms (SNPs) that substitute amino acids in the X-ray repair cross-complementing gene 1 (XRCC1) protein, Arg194Trp (R194W), Arg280His (R280H), and Arg399Gln (R399Q), to the risk of various types of cancers has been extensively investigated by epidemiological researches. To investigate whether two of these polymorphisms directly influence their repair ability, we established Chinese hamster ovary (CHO) EM9 cell lines transfected with XRCC1(WT), XRCC1(R194W), or XRCC1(R280H) genes and analyzed the DNA repair ability of these cells. The EM9 cells that lack functional XRCC1 proteins exhibit severe sensitivity to methyl methanesulfonate (MMS). Introduction of the human XRCC1(WT) and XRCC1(R194W) gene to EM9 cells restored the MMS sensitivity to the same level as the AA8 cells, a parental cell line. However, introduction of the XRCC1(R280H) gene partially restored the MMS sensitivity, resulting in a 1.7- to 1.9-fold higher sensitivity to MMS compared with XRCC1(WT) and XRCC1(R194W) cells at the LD(50) value. The alkaline comet assay determined diminished base excision repair/single strand break repair (BER/SSBR) efficiency in XRCC1(R280H) cells as observed in EM9 cells. In addition, the amount of intracellular NAD(P)H decreased in XRCC1(R280H) cells after MMS treatment. Indirect immunofluorescence staining of the XRCC1 protein showed an intense increase in the signals and clear foci of XRCC1 in the nuclei of the XRCC1(WT) cells, but a faint increase in the XRCC1(R280H) cells, after MMS exposure. These results suggest that the XRCC1(R280H) variant protein is defective in its efficient localization to a damaged site in the chromosome, thereby reducing the cellular BER/SSBR efficiency.