Abstract
Once double-strand breaks (DSBs) are generated within genome DNA, DSBs are immdiately recognized by sensor mechanisms and then activate ATM kinase for phosphorylation of the substrates, which leads to the cellular responses such as cell cycle checkpoints, apoptosis and DNA repair. Recently, it was reported that the C-terminus of NBS1 has an indispensable role for recruitment of ATM to DSB sites and possibly for the resulting phosphorylation of substrates through the interaction with ATM. Previously, we demonstrated that gamma-H2AX is involved in the recruitment of NBS1/hMre11/hRAD50 complex to DSB sites through interaction with NBS1 at the N-terminus. Hence, gamma-H2AX might function, as a sensor or initial factor, in ATM-dependent damage response. We showed here that gamma-H2AX forms the complex with ATM and NBS1 in irradiated cells. When the expression of H2AX was repressed by H2AX siRNA, chromatin-bound NBS1 in irradiated cells was significantly decreased. Early focus formation of NBS1 and phosphorylated ATM was also attenuated. As a result, H2AX siRNA reduced the phosphorylation of ATM substrates, which were present in gamma-H2AX complex in irradiated cells. However, these phosphorylations were restored a few hours after irradiation, indicating an altenative activation pathway for ATM-dependent damage response. Taken together, our results suggest that gamma-H2AX could mediate a rapid activation pathway of ATM through interaction with ATM and the substrates.
