PARENT SESSION
1:30 PM to 3:30 PM
Monday, April 22, 2002
Poster Session 15 DNA Damage and Repair II
Room: Nevada Exhibition Center

(P20-210) ATM Phosphorylates Histone H2AX in Response to DNA Double-Strand Breaks.

Burma, Sandeep^*,1, Chen, Benjamin¹, Murphy, Michael¹, Kurimasa, Akihiro², Chen, David¹, ¹ Lawrence Berkeley National Laboratory, Life Sciences Division, Berkeley, CA² Tottori University, Tottori, JAPAN

ABSTRACT-
A very early step in the response of mammalian cells to DNA double-strand breaks (DSBs) is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. While the PI-3 kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. In order to definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs-/- and Atm-/- cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs-/- cells and severely compromised in Atm-/- cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm-/- fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm-/- fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish 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 cellular response to DSBs. This work was funded by US Dept. of Energy and NIH grant CA50519 to D.J.C.

KEYWORDS: H2AX, ATM, DNA-PK, DNA double-strand breaks