Nazarov, I. B., Smirnova, A. N., Krutilina, R. I., Svetlova, M. P., Solovjeva, L. V., Nikiforov, A. A., Oei, S-L., Zalenskaya, I. A., Yau, P. M., Bradbury, E. M. and Tomilin, N. V. Dephosphorylation of Histone γ-H2AX during Repair of DNA Double-Strand Breaks in Mammalian Cells and its Inhibition by Calyculin A. Radiat. Res. 160, 309–317 (2003).

The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called γ-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that γ-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase. Here we studied the dynamics of dephosphorylation of γ-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of γ-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the γ-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, γ-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites. We also found that calyculin A inhibited early elimination of γ-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from γ-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and γ-H2AX and the coupling of its in situ dephosphorylation to DSB repair.