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 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.
Kumagai
Akiko
A
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Kim
Soo-Mi
SM
Dunphy
William G
WG
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010063:Ovum / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins / Q000378:metabolism*
C495984:ATRIP protein, Xenopus; C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; C416368:CLSPN protein, Xenopus; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D029867:Xenopus Proteins; D004247:DNA; D011494:Protein Kinases; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases; D005090:Exodeoxyribonucleases; C429526:three prime repair exonuclease 1