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

Radiat Res. 2003 Sep;160(3):309-17. doi: 10.1667/rr3043.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antimetabolites, Antineoplastic / pharmacology
  • Antineoplastic Agents, Alkylating / pharmacology
  • Bleomycin / pharmacology
  • Cell Nucleus / metabolism
  • Cells, Cultured
  • Chromatin / metabolism
  • Cricetinae
  • DNA Damage
  • DNA Repair
  • DNA-Binding Proteins / metabolism
  • Electrophoresis, Gel, Pulsed-Field
  • Fibroblasts / metabolism
  • Green Fluorescent Proteins
  • Histones / chemistry
  • Histones / metabolism*
  • Humans
  • Immunoblotting
  • Kinetics
  • Luminescent Proteins / metabolism
  • Marine Toxins
  • Methyl Methanesulfonate / pharmacology
  • Microscopy, Fluorescence
  • Oxazoles / pharmacology
  • Phosphoprotein Phosphatases / metabolism
  • Phosphorylation
  • Plasmids / metabolism
  • Protein Phosphatase 1
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae Proteins*
  • Serine / chemistry
  • Time Factors
  • Tumor Cells, Cultured
  • Ubiquitin-Protein Ligases


  • Antimetabolites, Antineoplastic
  • Antineoplastic Agents, Alkylating
  • Chromatin
  • DNA-Binding Proteins
  • H2AX protein, human
  • Histones
  • Luminescent Proteins
  • Marine Toxins
  • Oxazoles
  • RAD18 protein, S cerevisiae
  • RAD18 protein, human
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Bleomycin
  • Green Fluorescent Proteins
  • Serine
  • calyculin A
  • Methyl Methanesulfonate
  • Ubiquitin-Protein Ligases
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 1