Abstract
DNA double-strand breaks (DSBs) are the most lethal damage in the radiation-induced DNA damage and are subject to misrepair. In higher vertebrate cells, there are at least two major DSB repair pathways, namely non-homologous end-joining (NHEJ) and homologous recombination (HR). It has been identified many DSB repair proteins, which are engaged in NHEJ and/or HR.
Recently, imaging analysis of foci formation of DSB repair proteins, such as phosphorylated histone H2AX, is one of the available methods to elucidate the mechanisms of DSB repair. However, it is difficult to observe the initial response of DSB repair proteins on the site of DSBs induced by X-rays, because conventional X-ray machines cannot perform target irradiation. In this study, we irradiated the targeted part of cell nuclei using synchrotron X-ray microbeam system and observed the localization and phosphorylation of DSB repair proteins by immunofluorescence.
We first identified the induction of DSBs in the irradiated site using the phosphorylated histone H2AX specific antibody. The localization of DSB repair proteins was observed in normal human fibroblast MRC-5 cells irradiated with X-ray microbeam and then fixed 30 min after irradiation. Localization of NBS1 and phosphorylated DNA-PKcs and ATM on the site of target irradiation could be observed predictably, but we identified a few proteins, which have been reported nuclear foci formation after conventional X-rays irradiation, were not localized. The localization of cell cycle checkpoint proteins will be presented.
