Abstract
The ATR and Chk1 kinases are essential to maintain genomic integrity. ATR, with Claspin and the Rad9-Rad1-Hus1 complex, activates Chk1 after DNA damage. Chk1-mediated phosphorylation of the Cdc25A phosphatase is required for the mammalian S-phase checkpoint. Here, we show that during physiological S phase the regulation of the Chk1-Cdc25A pathway depends on ATR, Claspin, Rad9, and Hus1. Human cells with chemically or genetically ablated ATR showed inhibition of Chk1-dependent phosphorylation of Cdc25A, and they accumulated Cdc25A without external DNA damage. Furthermore, siRNA-mediated depletion of Claspin, Rad9 and Hus1 also stabilized Cdc25A. ATR ablation also inhibited the activatory phosphorylation of Chk1 on serine 345. Thus, the ATR-Chk1-Cdc25A pathway represents an integral part of physiological S-phase progression, and interference with this mechanism undermines viability of somatic mammalian cells. DNA damage further activates and switches this pathway from its constitutively operating "surveillance mode" compatible with DNA replication into an "emergency" checkpoint response.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism*
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Ataxia Telangiectasia Mutated Proteins
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Checkpoint Kinase 1
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DNA Damage / genetics
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DNA Replication / physiology
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Down-Regulation / physiology
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Exonucleases / genetics
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Exonucleases / metabolism
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Genomic Instability / physiology
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Humans
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Phosphorylation
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Protein Kinases / genetics
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Protein Kinases / metabolism*
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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RNA, Small Interfering / genetics
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RNA, Small Interfering / metabolism
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S Phase / physiology
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Schizosaccharomyces pombe Proteins / genetics
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Schizosaccharomyces pombe Proteins / metabolism
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Signal Transduction / physiology
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Tumor Cells, Cultured
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cdc25 Phosphatases / genetics
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cdc25 Phosphatases / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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CLSPN protein, human
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Cell Cycle Proteins
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RNA, Small Interfering
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Schizosaccharomyces pombe Proteins
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hus1 protein, S pombe
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rad9 protein
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Protein Kinases
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ATR protein, human
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Ataxia Telangiectasia Mutated Proteins
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CHEK1 protein, human
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Checkpoint Kinase 1
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Chk1 protein, S pombe
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Protein Serine-Threonine Kinases
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Exonucleases
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Rad1 protein, human
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CDC25C protein, human
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cdc25 Phosphatases