Abstract
TopBP1 and the Rad9-Rad1-Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR-ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-alpha to sites of replication stress. Furthermore, we show that pol-alpha is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-alpha, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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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 Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Chromatin / metabolism
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Chromosomal Proteins, Non-Histone / genetics
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Chromosomal Proteins, Non-Histone / metabolism*
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Cloning, Molecular
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DNA / metabolism*
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DNA Polymerase I / genetics
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DNA Polymerase I / metabolism*
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DNA Replication*
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DNA-Binding Proteins / metabolism
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Multiprotein Complexes
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Mutagenesis, Site-Directed
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Protein Serine-Threonine Kinases / metabolism
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Signal Transduction*
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Stress, Physiological
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Time Factors
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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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Chromatin
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Chromosomal Proteins, Non-Histone
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DNA-Binding Proteins
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Hus1 protein, Xenopus
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Multiprotein Complexes
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Rad1 protein, Xenopus
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TopBP1 protein, Xenopus
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Tumor Suppressor Proteins
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Xenopus Proteins
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rad9 protein
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DNA
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Atr protein, Xenopus
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Ataxia Telangiectasia Mutated Proteins
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Protein Serine-Threonine Kinases
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DNA Polymerase I