Abstract
A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate gamma-H2AX. Here, we demonstrate that gamma-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which gamma-H2AX forms does not spread as DSBs persist; rather, gamma-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate gamma-H2AX, only ATM promotes gamma-H2AX formation to maximal distance and maintains gamma-H2AX densities. MDC1 is essential for gamma-H2AX formation at high densities near DSBs, but not for generation of gamma-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of gamma-H2AX formed. Our data suggest that H2AX fuels a gamma-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing
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Animals
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Ataxia Telangiectasia Mutated Proteins
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B-Lymphocytes / cytology
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B-Lymphocytes / physiology
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Chromatin / metabolism*
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DNA / metabolism*
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DNA Damage*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Endonucleases
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G1 Phase / physiology
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Genes, T-Cell Receptor alpha / genetics
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Histones / genetics
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Histones / metabolism*
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism*
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Mice
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Mice, Knockout
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Recombination, Genetic
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Thymus Gland / cytology
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Tumor Suppressor Protein p53 / genetics
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Tumor Suppressor Protein p53 / metabolism
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Tumor Suppressor Proteins / genetics
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Tumor Suppressor Proteins / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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Cell Cycle Proteins
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Chromatin
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DNA-Binding Proteins
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H2AX protein, mouse
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Histones
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Intracellular Signaling Peptides and Proteins
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MDC1 protein, mouse
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Nuclear Proteins
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Tumor Suppressor Protein p53
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Tumor Suppressor Proteins
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DNA
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Ataxia Telangiectasia Mutated Proteins
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Atm protein, mouse
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Protein Serine-Threonine Kinases
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Endonucleases
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Dclre1c protein, mouse