Cell-cycle checkpoints induced by DNA damage or replication play critical roles in the maintenance of genomic integrity during cell proliferation. Biochemical analysis of checkpoint pathways has been greatly facilitated by the use of cell-free systems made from Xenopus eggs. In the present study, we describe a human cell-free system that reproduces a DNA-dependent checkpoint pathway acting on the Chk1 protein kinase. In this system, double-stranded DNA oligonucleotides induce the phosphorylation of Chk1 at activating sites targeted by ATR [ATM (ataxia telangiectasia mutated)- and Rad3-related] and ATM kinases. Phosphorylation of Chk1 is dependent on the interaction of Claspin, a protein first identified in Xenopus as a Chk1-binding protein. We show that the DNA-dependent binding of Chk1 to Claspin requires two phosphorylation sites, Thr916 and Ser945, which lie within the Chk1-binding domain of Claspin. Using a phosphopeptide derived from the consensus motif of these sites, we show that the interaction of Claspin with Chk1 is required for the ATR/ATM-dependent phosphorylation of Chk1. Using a panel of protein kinase inhibitors, we provide evidence that Chk1 is phosphorylated at an additional site in response to activation of the checkpoint response, probably by autophosphorylation. Claspin is phosphorylated in the Chk1-binding domain in an ATR/ATM-dependent manner and is also targeted by additional kinases in response to double-stranded DNA oligonucleotides. This cell-free system will facilitate further biochemical analysis of the Chk1 pathway in humans.
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Research Article|
May 24 2005
DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system
Catriona A. L. CLARKE;
Catriona A. L. CLARKE
1Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
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Paul R. CLARKE
Paul R. CLARKE
1
1Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
1To whom correspondence should be addressed (email paul.clarke@cancer.org.uk).
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Publisher: Portland Press Ltd
Received:
November 25 2004
Revision Received:
January 20 2005
Accepted:
February 14 2005
Accepted Manuscript online:
February 14 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 388 (2): 705–712.
Article history
Received:
November 25 2004
Revision Received:
January 20 2005
Accepted:
February 14 2005
Accepted Manuscript online:
February 14 2005
Citation
Catriona A. L. CLARKE, Paul R. CLARKE; DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system. Biochem J 1 June 2005; 388 (2): 705–712. doi: https://doi.org/10.1042/BJ20041966
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