Abstract
The repair of DNA double-strand breaks (DSB) requires processing of the broken ends to complete the ligation process. Nonhomologous end-joining (NHEJ) is the main repair mechanism of DSBs in mammalian cells. DNA polymerase mu(pol mu) and pol lambda are members of the mammalian pol X family of DNA polymerases and both polymerases form a specific complex with the NHEJ core factors Ku and XRCC4-LigIV, implicating them as end-processing proteins in NHEJ. Recent studies have shown that the method of DSB induction by laser micro-irradiation can be used to study NHEJ in vivo. The early response of NHEJ factors to the onset of DSBs has been shown using live cell imaging, which indicated that they are recruited to laser-induced DSBs in a matter of seconds. However, the accumulation mechanisms of pol mu at DSBs have not been clarified in vivo. In this study, we examined the accumulation mechanism of pol mu at DSBs in living cells. Pol mu accumulation at DSBs began immediately after irradiation. Also the deletion mutants of pol mu retained their accumulation activity, although the mutants partly reduced their accumulation activity. These data demonstrate that at least three domains of pol mu, i.e., the N-terminal BRCT domain, the DNA-binding HhH motif, and the C-teminal domain, seem to necessary for the accumulation at DSBs in the early stage after irradiation. Furthermore, by the use of photobleaching and photoconversion techniques, we found that pol mu exchanges within a few seconds between the bound state in the repair complex and the diffusive state in the nucleoplasm.
