Abstract
Chromosomal abnormalities are frequently caused by problems encountered during DNA replication. Although the ATR-Chk1 pathway has previously been implicated in preventing the collapse of stalled replication forks into double-strand breaks (DSB), the importance of the response to fork collapse in ATR-deficient cells has not been well characterized. Herein, we demonstrate that, upon stalled replication, ATR deficiency leads to the phosphorylation of H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of homologous recombination and fork restart. Because H2AX has been shown to play a facilitative role in homologous recombination, we hypothesized that H2AX participates in Rad51-mediated suppression of DSBs generated in the absence of ATR. Consistent with this model, increased Rad51 focal accumulation in ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR and H2AX lead to synergistic increases in chromatid breaks and translocations. Importantly, the ATM and DNA-PK phosphorylation site on H2AX (Ser(139)) is required for genome stabilization in the absence of ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal role in suppressing DSBs during DNA synthesis in instances of ATR pathway failure. These results imply that ATR-dependent fork stabilization and H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress double-strand breaks as a layered response network when replication stalls.
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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Animals
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Ataxia Telangiectasia Mutated Proteins
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Cell Cycle Proteins / antagonists & inhibitors
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Cell Cycle Proteins / metabolism
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Cell Cycle Proteins / physiology*
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Cells, Cultured
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DNA Breaks, Double-Stranded*
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DNA Replication*
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DNA-Activated Protein Kinase / metabolism
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DNA-Binding Proteins / metabolism
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Embryo, Mammalian / cytology
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Embryo, Mammalian / metabolism
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Embryo, Mammalian / radiation effects
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Fibroblasts / cytology
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Fibroblasts / metabolism
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Fibroblasts / radiation effects
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Genomic Instability*
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Histones / physiology*
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Metaphase
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Mice
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Mice, Knockout
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Mitosis
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Nuclear Proteins / metabolism
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Phosphorylation
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / metabolism
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Protein Serine-Threonine Kinases / physiology*
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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RNA, Small Interfering / pharmacology
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Rad51 Recombinase / metabolism
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Radiation, Ionizing
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Reverse Transcriptase Polymerase Chain Reaction
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S Phase / physiology
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Spectral Karyotyping
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Tumor Suppressor Proteins / metabolism
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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H2AX protein, mouse
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Histones
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Nuclear Proteins
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RNA, Messenger
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RNA, Small Interfering
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Tumor Suppressor Proteins
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Atr protein, mouse
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Ataxia Telangiectasia Mutated Proteins
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Atm protein, mouse
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DNA-Activated Protein Kinase
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Prkdc protein, mouse
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Protein Serine-Threonine Kinases
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Rad51 Recombinase
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Rad51 protein, mouse