Dephosphorylation of Histone γ-H2AX during Repair of DNA Double-Strand Breaks in Mammalian Cells and its Inhibition by Calyculin A

Abstract

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 gamma-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that gamma-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 gamma-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of gamma-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the gamma-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, gamma-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 gamma-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from gamma-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and gamma-H2AX and the coupling of its in situ dephosphorylation to DSB repair.

Replication stress activates DNA polymerase alpha-associated Chk1

“…Depletion of Polα was associated with a mild accumulation of dsDNA breaks, assessed by intracellular of γ-H2A.X staining and FACS. 42,43 Of note, γ-H2A.X was detected in 3N populations, suggestive of DNA damage within cells traversing S-phase (Fig. 1C).…”

3-Methyladenine DNA glycosylase is important for cellular resistance to psoralen interstrand cross-links

“…2, panels A and B; Supplemental Figure 3A). UVA induction of γ-H2AX foci formation was previously shown [44] and is probably the consequence of oxidized DNA lesions [45,46] creating sites of single strand DNA and/or stalled replication forks that could also be sites for γ-H2AX foci formation [47][48][49][50]. UVA treated wild-type and Aag−/− cell cultures had many cells with 11-30 foci per cell by 6 hours after treatment (data not shown) but very few cells with >50 foci per cell (12% for both wild-type and Aag−/−; Fig.…”

TFII-I: Connecting Mitogenic Signals to Cell Cycle Regulation

“…Likewise, TFII-I knockdown cells also exhibit accelerated reduction of γ-H2AX following IR-induced DNA damage (Desgranges ZP, Roy AL, unpublished). The observation that TFII-I influences persistence of γ-H2AX foci and thus affects DSB repair 49 suggests a role for TFII-I in DNA repair. An alternative possibility is that TFII-I influences the phosphorylation status of H2AX without directly affecting DNA repair.…”