Claspin and the Activated Form of ATR-ATRIP Collaborate in the Activation of Chk1

Claspin is necessary for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. ATR possesses a regulatory partner called ATRIP. We have studied the respective roles of ATR-ATRIP and Claspin in the activation of Chk1. ATR-ATRIP bound well to various DNA...

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Bibliographic Details
Published in:The Journal of biological chemistry 2004-11, Vol.279 (48), p.49599-49608
Main Authors: Kumagai, Akiko, Kim, Soo-Mi, Dunphy, William G.
Format: Article
Language:English
Subjects:
Adaptor Proteins, Signal Transducing - metabolism
Amino Acid Sequence
Animals
Ataxia Telangiectasia Mutated Proteins
Carrier Proteins - metabolism
Cell Cycle Proteins - metabolism
Checkpoint Kinase 1
DNA - metabolism
DNA-Binding Proteins
Exodeoxyribonucleases - metabolism
Humans
Molecular Sequence Data
Ovum - metabolism
Phosphoproteins - metabolism
Protein Kinases - metabolism
Protein-Serine-Threonine Kinases - metabolism
Xenopus
Xenopus Proteins - metabolism
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Summary:Claspin is necessary for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. ATR possesses a regulatory partner called ATRIP. We have studied the respective roles of ATR-ATRIP and Claspin in the activation of Chk1. ATR-ATRIP bound well to various DNA templates in Xenopus egg extracts. ATR-ATRIP bound to a single-stranded DNA template was weakly active. By contrast, the ATR-ATRIP complex on a DNA template containing both single- and double-stranded regions displayed a large increase in kinase activity. This observation suggests that ATR-ATRIP normally undergoes activation upon association with specific nucleic acid structures at DNA replication forks. Without Claspin, activated ATR-ATRIP phosphorylated Chk1 weakly in a cell-free reaction. The addition of Claspin to this reaction strongly stimulated the phosphorylation of Chk1 by ATR-ATRIP. Claspin also induced significant autophosphorylation of Chk1 in the absence of ATR-ATRIP. Taken together, these results indicate that the checkpoint-dependent phosphorylation of Chk1 is a multistep process involving activation of the ATR-ATRIP complex at replication forks and presentation of Chk1 to this complex by Claspin.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M408353200