Abstract
DNA double-strand breaks (DSB) repair requires the processing of the broken DNA ends to accomplish the ligation. Non-homologous end-joining (NHEJ) is a main mechanism of DSB repairs in mammalian cells. DNA polymerases mu (pol mu) and lambda (pol lambda) belong to mammalian pol X family members and form a specific complex with Ku and XRCC4-LigIV, which are essential for DNA end-processing in NHEJ. The live cell imaging has shown that these NHEJ factors rapidly recruit to the DSBs. However, it has not been clarified how pol mu recruits to the DSBs in vivo. We report here the detailed mechanism of pol mu's recruitment to the DSBs.
We first observed pol mu at DSBs in living cells after laser micro-irradiation. pol mu accumulated at DSBs immediately after irradiation. By photo-bleaching and photo-conversion techniques, we found that pol mu exchanges between the DSBs and the nucleoplasm within a few seconds. Furthermore, pol mu accumulated at DSBs independently of Ku80. These observations are quite different from those of Ku80-interacting NHEJ core factors, i.e., Ku70, DNA-PKcs (DNA-dependent protein kinase), XRCC4, and XLF (XRCC4-like factor), which Ku80-dependently accumulate at DSBs. Whereas the N-terminal region of pol mu containing BRCT domain, which binds to Ku80, Ku80-dependently accumulated at DSBs, the C-terminal pol beta-like region of pol mu Ku80-independently accumulated at DSBs. The pol beta-like region of pol mu contains DNA-binding motif HhH (Helix-hairpin-Helix) and PCNA-interacting motif (PIM)-like region. Both of them retained the ability of accumulating at DSBs. Biochemical analyses showed that gamma-ray irradiation stimulates pol mu-PCNA interaction and the interaction is sensitive to ethidium bromide, suggesting that dsDNA affects on the pol mu-PCNA interaction.
Our studies show that pol mu recruites to DSBs at the first step during the NHEJ process and three functional domains in pol mu individually have abilities to accumulate at DSBs after irradiation.
