Dephosphorylation of histone γ-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A

I. B. Nazarov, A. N. Smirnova, R. I. Krutilina, M. P. Svetlova, L. V. Solovjeva, A. A. Nikiforov, S. L. Oei, I. A. Zalenskaya, P. M. Yau, E. M. Bradbury, N. V. Tomilin

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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 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.

Original languageEnglish
Pages (from-to)309-317
Number of pages9
JournalRadiation Research
Volume160
Issue number3
DOIs
Publication statusPublished - Sep 1 2003

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Radiology Nuclear Medicine and imaging
  • Biophysics
  • Radiation

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    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., & Tomilin, N. V. (2003). Dephosphorylation of histone γ-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A. Radiation Research, 160(3), 309-317. https://doi.org/10.1667/RR3043