Abstract
DNA double-strand breaks (DSBs) are the most serious threat generated by ionizing radiation (IR). There are two primary pathways for DSB repair in mammalian cells, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR occurs only in the late S to G2 phases of cell cycle, while NHEJ can function throughout the cell cycle. XRCC4, which was isolated as a factor complementing the radiosensitive phenotype of a mutant rodent cell line, is known as a core factor of NHEJ. We have generated XRCC4-deficient (XRCC4-/-) cells from the human colon adenocarcinoma cell line HCT116 by gene targeting. The human XRCC4-/- cells were insufficient for DSB repair and hypersensitive to X-irradiation. On the other had, analysis of sister chromatid exchange revealed that the XRCC4-/- cells were sufficient to HR. Thus, the XRCC4-/- cells are NHEJ-deficient. To elucidate the contribution of reactive oxygen species to the generation of DNA damage elicited by IR, we examined the cellular responses of the XRCC4-/- cells to hydrogen peroxide (H2O2). The XRCC4-/- cells exhibited higher sensitivity to H2O2 than HCT116 cells by a survival assay. Formation of γH2AX foci, a decision marker of DSBs, was observed in both cell lines exposed to H2O2. Interestingly, most of the γH2AX foci induced by H2O2 did not colocalized with the ATM foci in contrast to those formed in cells exposed to IR. The kinetic analysis of chromosomal aberrations after the insults demonstrated that the XRCC4-/- cells were highly susceptible to H2O2 during the late G1 to early S phases. It is probable that DNA damages induced by H2O2 may be converted to DSBs during DNA replication and those DSBs must be repaired not only by HR but also by NHEJ.
