title: Function of a conserved checkpoint recruitment domain in ATRIP proteins.
abstract: The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling. Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP.
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Ball
authors.forename: Mark R
authors.initials: MR
authors.lastname: Ehrhardt
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Mordes
authors.forename: Gloria G
authors.initials: GG
authors.lastname: Glick
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
authors.forename: David
authors.initials: D
authors.lastname: Cortez
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008279:Magnetic Resonance Imaging; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C440014:LCD1 protein, S cerevisiae; D010750:Phosphoproteins; C082728:RFA1 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D005090:Exodeoxyribonucleases; C429526:three prime repair exonuclease 1