title: ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable for Chk1 phosphorylation.
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.
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Ball
authors.forename: Jeremy S
authors.initials: JS
authors.lastname: Myers
authors.forename: David
authors.initials: D
authors.lastname: Cortez
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000235:genetics; D051716:Replication Protein A; D015398:Signal Transduction; D014162:Transfection; D020798:Two-Hybrid System Techniques
chemical_list: C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C497167:RPA1 protein, human; D051716:Replication Protein A; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D005090:Exodeoxyribonucleases; C429526:three prime repair exonuclease 1
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