Abstract
Among various types of DNA damages, DNA double-strand breaks (DSBs) are considered most critical determinant of the fate of the cells or organisms exposed to radiation. DSBs are repaired mainly through two pathways; non-homologous end joining (NHEJ) and homologous recombination. XRCC4 association with DNA-LigaseIV is considered one of pivotal molecule in NHEJ pathway. We have demonstrated that XRCC4 undergoes is phosphorylated by DNA-PK in response to radiation, although biological significance of phosphorylation remains to be elucidated. In this study, we examine the effect of nuclear reactor irradiation which contains neutron and gamma ray.
We used murine leukemia L5178Y-derived, XRCC4-deficient cell line M10 as the host and introduced empty pCMV10 vector (M10-CMV), normal XRCC4 cDNA (M10-XRCC4) or mutant XRCC4 cDNA. Cells were irradiated in Nuclear reactor UTR-KINKI at Kinki University. Dose rate of neutron was 19 cGy/h and that of gamma ray was 19 cGy/h.
Radiosensitivity was evaluated by colony forming ability in media containing 0.16 % agarose. To measure mutation frequency at HPRT (hypoxanthine phosphoribosyl transferase) locus cell were plated in media at containing agarose with 5 ng/ml of 6-thioguanine (6-TG). The fraction of mutant was increased by approximately 6 - 20 folds in multiple experiments.
The mutant could not be recovered from irradiated M10-CMV. This frequency would be equivalent to 10 Gy of X-ray according to an earlier study by others on L5178Y (Shiomi et al., 1981). These results suggested that DNA damage induced by neutron might be hard to be repaired correctly and NHEJ repair of neutron-induced DNA damage might result in incorrect repair causing mutation. Further studies are ongoing to examine effect of DNA-PK inhibitors on mutation frequency and to analyze cells expressing mutant XRCC4 lacking phosphorylation sites.
