ATM phosphorylates histone H2AX in response to DNA double-strand breaks

A very early step in the response of mammalian cells to DNA double-strand b reaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-depend ent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specif ic kinase involved has not yet been identified. To definitively identify th e specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA -PKcs -/- and Atm-/- cell lines and mouse embryonic fibroblasts. We find th at H2AX phosphorylation and nuclear focus formation are normal in DNA-PKes- /- cells and severely compromised in Atm-/- cells. We also find that ATM ca n phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm-/- fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phospho rylation in Atm-/- fibroblasts can be abolished by low concentrations of wo rtmannin suggesting that DNA-PK, rather than ATR, is responsible for low le vels of H2AX phosphorylation in the absence of ATM. Our results clearly est ablish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cell ular response to double-strand breaks.