Host: The Japan Radiation Research Society, Chairman of the 52nd Annual Meeting, Toshiteru Okubo (Radiation Effects Research Foundation)
Purpose: XRCC4, in association with DNA ligase IV and XLF, is necessary for the ligation of the two DNA ends, as the final step of DNA double-strand break repair through non-homologous end-joining. It is shown that XRCC4 is phosphorylated in vitro and in vivo by DNA-PK, which is considered the molecular sensor of DNA double-strand breaks. We have so far identified four phosphorylation sites, in addition to two identified by others. Among four phosphorylation sites, two were phosphorylated in cellulo, i.e., in murine leukemia M10-derived XRCC4 expressing cell lines. On the other hand, we have been unable to detect the phosphorylation of other two sites. Considering a possible difference in the manner of phosphorylation between human and rodent cells, we examined phosphorylation status of XRCC4 in human cells.
Methods: We collected XRCC4 protein from 0.5L culture of human leukemia MOLT-4 cells, either left unirradiated or harvested 30min after 20Gy 60Co gamma-irradiation), by immunoaffinity column chromatography. The phosphorylation status was analyzed by Western blotting using phosphorylation-specific antibodies corresponding to respective sites.
Results and discussion: We could detect the phosphorylation of all of four phosphorylation sites. The phosphorylation was enhanced after irradiation. This observation indicated that the manner of phosphorylation of XRCC4 in response to DNA damage is considerably different between human and murine cell lines. We will also report a new, rapid assay system to evaluate XRCC4 function in terms of its ability to sustain proliferative capacity after irradiation.
