Abstract
ATR associates with the regulatory protein ATRIP that has been proposed to localize ATR to sites of DNA damage through an interaction with single-stranded DNA (ssDNA) coated with replication protein A (RPA). We tested this hypothesis and found that ATRIP is required for ATR accumulation at intranuclear foci induced by DNA damage. A domain at the N terminus of ATRIP is necessary and sufficient for interaction with RPA-ssDNA. Deletion of the ssDNA-RPA interaction domain of ATRIP greatly diminished accumulation of ATRIP into foci. However, the ATRIP-RPA-ssDNA interaction is not sufficient for ATRIP recognition of DNA damage. A splice variant of ATRIP that cannot bind to ATR revealed that ATR association is also essential for proper ATRIP localization. Furthermore, the ATRIP-RPA-ssDNA interaction is not absolutely essential for ATR activation because ATR phosphorylates Chk1 in cells expressing only a mutant of ATRIP that does not bind to RPA-ssDNA. These data suggest that binding to RPA-ssDNA is not the essential function of ATRIP in ATR-dependent checkpoint signaling and ATR has an important function in properly localizing the ATR-ATRIP complex.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Signal Transducing
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Ataxia Telangiectasia Mutated Proteins
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Base Sequence
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Binding Sites / genetics
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Cell Cycle Proteins / metabolism*
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Cell Line
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Cell Nucleus / metabolism
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Checkpoint Kinase 1
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DNA Damage
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DNA, Single-Stranded / metabolism*
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DNA-Binding Proteins / metabolism*
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Exodeoxyribonucleases / chemistry
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Exodeoxyribonucleases / genetics
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Exodeoxyribonucleases / metabolism*
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Humans
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In Vitro Techniques
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Phosphoproteins / chemistry
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Phosphoproteins / genetics
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Phosphoproteins / metabolism*
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Phosphorylation
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Protein Binding
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Protein Kinases / metabolism*
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Protein Serine-Threonine Kinases / metabolism*
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Protein Structure, Tertiary
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RNA, Small Interfering / genetics
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Replication Protein A
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Signal Transduction
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Transfection
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Two-Hybrid System Techniques
Substances
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ATRIP protein, human
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Adaptor Proteins, Signal Transducing
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Cell Cycle Proteins
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DNA, Single-Stranded
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DNA-Binding Proteins
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Phosphoproteins
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RNA, Small Interfering
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RPA1 protein, human
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Replication Protein A
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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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Exodeoxyribonucleases
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three prime repair exonuclease 1