Abstract
Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AX(S136A/S139A)) indicate that phosphorylated H2AX (gammaH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to gammaH2AX is BRCA1-dependent and cells deficient or depleted of H2AX show an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX-deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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BRCA1 Protein / genetics
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BRCA1 Protein / metabolism
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Cell Cycle / drug effects
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Cell Cycle / genetics
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Cell Cycle / radiation effects
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Cell Line
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Cell Line, Tumor
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Cell Survival / drug effects
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Cell Survival / genetics
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Cell Survival / radiation effects
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Chromosomal Instability / drug effects
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Chromosomal Instability / radiation effects
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DNA Damage
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DNA Replication / drug effects
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DNA Replication / genetics*
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DNA Replication / radiation effects
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Fanconi Anemia Complementation Group D2 Protein / genetics*
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Fanconi Anemia Complementation Group D2 Protein / metabolism
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Fanconi Anemia Complementation Group D2 Protein / physiology
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Fanconi Anemia Complementation Group Proteins / genetics
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Fanconi Anemia Complementation Group Proteins / metabolism
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Flow Cytometry
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HeLa Cells
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Histones / genetics*
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Histones / metabolism
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Histones / physiology
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Humans
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Immunohistochemistry
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Immunoprecipitation
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Mice
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Mice, Knockout
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Mitomycin / pharmacology
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Nucleic Acid Synthesis Inhibitors / pharmacology
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Signal Transduction / drug effects
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Signal Transduction / genetics
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Signal Transduction / radiation effects
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Ultraviolet Rays
Substances
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BRCA1 Protein
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Fanconi Anemia Complementation Group D2 Protein
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Fanconi Anemia Complementation Group Proteins
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H2AX protein, human
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Histones
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Nucleic Acid Synthesis Inhibitors
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Mitomycin