Abstract
ATR is a key regulator of checkpoint responses to incompletely replicated and damaged DNA, but the mechanisms underlying control of its kinase activity are unknown. TopBP1, the vertebrate homolog of yeast Cut5/Dbp11, has dual roles in initiation of DNA replication and regulation of checkpoint responses. We show that recombinant TopBP1 induces a large increase in the kinase activity of both Xenopus and human ATR. The ATR-activating domain resides in a conserved segment of TopBP1 that is distinct from its numerous BRCT repeats. The isolated ATR-activating domain from TopBP1 induces ectopic activation of ATR-dependent signaling in both Xenopus egg extracts and human cells. Furthermore, Xenopus egg extracts containing a version of TopBP1 with an inactivating point mutation in the ATR-activating domain are defective in checkpoint regulation. These studies establish that activation of ATR by TopBP1 is a crucial step in the initiation of ATR-dependent signaling processes.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Amino Acid Sequence
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Animals
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Ataxia Telangiectasia Mutated Proteins
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Cycle / physiology*
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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DNA Damage
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DNA Repair
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DNA-Binding Proteins / metabolism
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Enzyme Activation
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Genes, Reporter
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Humans
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Macromolecular Substances
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Molecular Sequence Data
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Nuclear Proteins
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Oocytes / physiology
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Point Mutation
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Protein Structure, Tertiary
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Sequence Alignment
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Signal Transduction / physiology
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Tumor Suppressor Proteins / metabolism
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Xenopus Proteins / genetics
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Xenopus Proteins / metabolism*
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Xenopus laevis
Substances
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ATRIP protein, Xenopus
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Carrier Proteins
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Cell Cycle Proteins
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DNA-Binding Proteins
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Macromolecular Substances
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Nuclear Proteins
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Recombinant Proteins
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TOPBP1 protein, human
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TopBP1 protein, Xenopus
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Tumor Suppressor Proteins
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Xenopus Proteins
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Atr protein, Xenopus
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ATM protein, human
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ATR protein, human
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Ataxia Telangiectasia Mutated Proteins
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Protein Serine-Threonine Kinases