DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system

Catriona A.L. Clarke, Paul R. Clarke (Lead / Corresponding author)

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85 Citations (Scopus)


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 aphosphopeptide 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.

Original languageEnglish
Pages (from-to)705-712
Number of pages8
JournalBiochemical Journal
Issue number2
Early online date1 Jun 2005
Publication statusPublished - 1 Jun 2005


  • Cell cycle
  • Checkpoint
  • Chk1
  • Claspin
  • DNA damage
  • Protein kinase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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