Association of MDC1 with non-homologous end-joining in DNA-damage response.

DNA double strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation (IR) and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) systems. Cells have invested in at least two pathways to repair DSBs, namely homologous recombination repair (HRR) and non-homologous end-joining (NHEJ). In higher organisms, NHEJ can function in all phases of the cell cycle and is the predominant repair pathway. We have established three cell lines having XRCC4, Artemis and MDC1 disrupted, respectively, by a gene targeting technique in a human colon tumor cell line HCT116 to define the biological roles of NHEJ-related genes on DNA damage induced by IR. Radio-sensitivities assessed by the clonogenic survival rate after X-ray exposure were apparently increased in these cell lines in comparison with the parental HCT116. Chromosomal aberrations induced by X-ray irradiation elevated significantly in three cell lines than in parental HCT116. X-ray-induced mutation frequency of the HPRT gene in MDC1-deficient cells was significantly higher than that in parental HCT116. These radiosensitive phenotypes might be due to insufficiency of DNA damage signaling/repair machinery in these deficient cell lines. In addition, NHEJ was impaired in MDC1- and XRCC4-deficient cells as assessed by random integration of plasmid DNA into the genome of these mutant cells. On the other hand, discrete foci of 53BP1, ATM (S1981), DNA-PKcs (S2056) and MDC1 formed in response to X-rays mostly co-localized to g-H2AX foci, a marker of DNA DSBs, in the nucleus of the parental HCT116. The formations of 53BP1and ATM (S1981) foci, however, were not induced by X-ray exposure in MDC1 deficient cells although positive immunoreactivities with specific antibodies were clearly detected throughout the nuclei of the deficient cells. Furthermore, foci of DNA-PKcs (S2056), relatively smaller than g-H2AX foci, were formed after X-ray irradiation in MDC1 deficient cells, but those foci did not co-localize to g-H2AX foci. These results suggest that MDC1 may be associated with the recruitment of DNA damage signaling/repair components, such as ATM, DNA-PKcs and 53BP1, to sites of DNA DSBs induced by IR, and that MDC1 might be the master regulator determining the formation of a specific chromatin microenvironment required for genomic stability. Finally, these mutant cell lines established will be useful for clarifying the mechanisms of response to DNA damage, since these possess an identical human genetic background.The 6th International Open Laboratory Workshop "Recent Advances in DNA Repair and Related Studies