Abstract
In eukaryotic cells, including mammal, DNA double strand breaks (DSBs) are repaired mainly through two pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA-PK complex, composed of DNA-PKcs, Ku86 and Ku70, and XRCC4/DNA ligase IV complex have been considered key molecules in NHEJ but new molecule, XLF/Cernunnos was found very recently. It is largely unknown how these proteins are recruited to DSB sites and assembled into repair machinery. Another mystery is the role of protein phosphorylating function of DNA-PK. For our understanding of DSB repair mechanism through NHEJ, it is critically important i) to visualize and analyze the recruitment process of NHEJ molecules to DSBs and ii) to elucidate the genuine target of DNA-PKcs and the physiological significance of phosphorylation. Regarding this, we have reported the detection and characterization of DNA-PKcs foci induced by ionizing radiation and, this time, we successfully detected the binding of XRCC4 to damaged chromatin DNA. Additionally, we have identified the phosphorylation site of XRCC4 by DNA-PKcs, which is phosphorylated in living cells after irradiation and disruption of which resulted in elevated radiosensitivity with partially impaired DNA repair capability. Based on these results, the process of recruitment and assembly of NHEJ machinery and the role of DNA-PK phosphorylation therein will be discussed.
