title: Dephosphorylation of histone gamma-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.
authors.affiliation: Department of Biological Chemistry, University of California Davis School of Medicine, Davis, California 95616, USA.
authors.forename: I B
authors.initials: IB
authors.lastname: Nazarov
authors.forename: A N
authors.initials: AN
authors.lastname: Smirnova
authors.forename: R I
authors.initials: RI
authors.lastname: Krutilina
authors.forename: M P
authors.initials: MP
authors.lastname: Svetlova
authors.forename: L V
authors.initials: LV
authors.lastname: Solovjeva
authors.forename: A A
authors.initials: AA
authors.lastname: Nikiforov
authors.forename: S-L
authors.initials: SL
authors.lastname: Oei
authors.forename: I A
authors.initials: IA
authors.lastname: Zalenskaya
authors.forename: P M
authors.initials: PM
authors.lastname: Yau
authors.forename: E M
authors.initials: EM
authors.lastname: Bradbury
authors.forename: N V
authors.initials: NV
authors.lastname: Tomilin
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D006224:Cricetinae; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008387:Marine Toxins; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D054645:Protein Phosphatase 1; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases
chemical_list: D000964:Antimetabolites, Antineoplastic; D018906:Antineoplastic Agents, Alkylating; D002843:Chromatin; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D008164:Luminescent Proteins; D008387:Marine Toxins; D010080:Oxazoles; C063607:RAD18 protein, S cerevisiae; C485954:RAD18 protein, human; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D001761:Bleomycin; D049452:Green Fluorescent Proteins; D012694:Serine; C059041:calyculin A; D008741:Methyl Methanesulfonate; D044767:Ubiquitin-Protein Ligases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1