Abstract
The ATR-mediated checkpoint is not only critical for responding to genotoxic stress but also essential for cell proliferation. The RFC-related checkpoint protein Rad17, a phosphorylation substrate of ATR, is critical for ATR-mediated checkpoint signaling and cell survival. Here, we show that phosphorylation of Rad17 by ATR is important for genomic stability and restraint of S phase but is not essential for cell survival. The phosphomutant Rad17AA exhibits distinct defects in hydroxyurea- (HU) and ultraviolet- (UV) induced Chk1 activation, indicating that separate Rad17 functions are required differently in response to different types of replication interference. Although cells expressing Rad17AA can initiate Chk1 phosphorylation after HU treatment, they fail to sustain Chk1 phosphorylation after withdrawal of HU and are profoundly sensitive to HU. Importantly, we found that phosphorylated Rad17 interacts with Claspin and regulates its phosphorylation. These findings reveal a phosphorylation-dependent function of Rad17 in an ATR-Rad17-Claspin-Chk1-signaling cascade that responds to specific replication stress.
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 / genetics
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Adaptor Proteins, Signal Transducing / metabolism*
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Ataxia Telangiectasia Mutated Proteins
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Cell Cycle / drug effects
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Cell Cycle / physiology
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Cell Cycle / radiation effects
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / physiology*
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Cell Line
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Cell Proliferation
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Cell Survival / drug effects
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Cell Survival / physiology
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Cell Survival / radiation effects
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Checkpoint Kinase 1
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Chromatin / metabolism
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DNA Damage / physiology
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DNA Replication / drug effects
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DNA Replication / physiology*
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DNA Replication / radiation effects
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Genomic Instability / physiology
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HCT116 Cells
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Histones / metabolism
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Humans
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Hydroxyurea / pharmacology
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Mutation / genetics
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Mutation / physiology
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Phosphorylation / drug effects
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Phosphorylation / radiation effects
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Protein Binding / drug effects
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Protein Binding / physiology
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Protein Binding / radiation effects
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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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S Phase / drug effects
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S Phase / physiology
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S Phase / radiation effects
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Transfection
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Ultraviolet Rays
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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Chromatin
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Histones
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Rad17 protein, human
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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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Protein Serine-Threonine Kinases
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Hydroxyurea