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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Antimetabolites, Antineoplastic / pharmacology
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Antineoplastic Agents, Alkylating / pharmacology
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Bleomycin / pharmacology
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Cell Nucleus / metabolism
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Cells, Cultured
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Chromatin / metabolism
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Cricetinae
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DNA Damage
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DNA Repair
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DNA-Binding Proteins / metabolism
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Electrophoresis, Gel, Pulsed-Field
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Fibroblasts / metabolism
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Green Fluorescent Proteins
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Histones / chemistry
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Histones / metabolism*
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Humans
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Immunoblotting
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Kinetics
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Luminescent Proteins / metabolism
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Marine Toxins
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Methyl Methanesulfonate / pharmacology
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Microscopy, Fluorescence
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Oxazoles / pharmacology
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Phosphoprotein Phosphatases / metabolism
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Phosphorylation
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Plasmids / metabolism
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Protein Phosphatase 1
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Recombinant Fusion Proteins / metabolism
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Saccharomyces cerevisiae Proteins*
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Serine / chemistry
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Time Factors
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Tumor Cells, Cultured
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Ubiquitin-Protein Ligases
Substances
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Antimetabolites, Antineoplastic
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Antineoplastic Agents, Alkylating
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Chromatin
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DNA-Binding Proteins
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H2AX protein, human
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Histones
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Luminescent Proteins
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Marine Toxins
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Oxazoles
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RAD18 protein, S cerevisiae
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RAD18 protein, human
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae Proteins
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Bleomycin
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Green Fluorescent Proteins
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Serine
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calyculin A
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Methyl Methanesulfonate
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Ubiquitin-Protein Ligases
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Phosphoprotein Phosphatases
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Protein Phosphatase 1