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.
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
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Amino Acid Sequence
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Animals
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Binding Sites
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Cell Cycle Proteins / chemistry
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Cycle*
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DNA Damage
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DNA, Fungal / genetics
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DNA-Binding Proteins / genetics
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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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Magnetic Resonance Imaging
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Models, Molecular
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Molecular Sequence Data
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Phosphoproteins / chemistry
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Phosphoproteins / genetics
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Phosphoproteins / metabolism*
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Protein Binding
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Protein Structure, Tertiary
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Replication Protein A / genetics
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Replication Protein A / metabolism
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Sequence Alignment
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Structural Homology, Protein
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Transcription Factors / genetics
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Transcription Factors / metabolism
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, Fungal
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DNA-Binding Proteins
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LCD1 protein, S cerevisiae
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Phosphoproteins
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RFA1 protein, S cerevisiae
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Replication Protein A
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Saccharomyces cerevisiae Proteins
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Transcription Factors
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Exodeoxyribonucleases
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three prime repair exonuclease 1