Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts

  1. Zijian Guo,
  2. Akiko Kumagai,
  3. Sophie X. Wang, and
  4. William G. Dunphy1
  1. Division of Biology, 216-76, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA

Abstract

The checkpoint kinase Xchk1 becomes phosphorylated inXenopus egg extracts in response to DNA replication blocks or UV-damaged DNA. Xchk1 is also required for the cell cycle delay that is induced by unreplicated or UV-damaged DNA. In this report, we have removed the Xenopus homolog of ATR (Xatr) from egg extracts by immunodepletion. In Xatr-depleted extracts, the checkpoint-associated phosphorylation of Xchk1 is abolished, and the cell cycle delay induced by replication blocks is strongly compromised. Xatr from egg extracts phosphorylated recombinant Xchk1 in vitro, but not a mutant form of Xchk1 (Xchk1-4AQ) containing nonphosphorylatable residues in its four conserved SQ/TQ motifs. Recombinant human ATR, but not a kinase-inactive mutant, phosphorylated the same sites in Xchk1. Furthermore, the Xchk1-4AQ mutant was found to be defective in mediating a checkpoint response in egg extracts. These findings suggest that Xchk1 is a functionally important target of Xatr during a checkpoint response to unreplicated or UV-damaged DNA.

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Footnotes

  • 1 Corresponding author.

  • E-MAIL dunphy{at}cco.caltech.edu; FAX (626) 795-7563.

  • Article and publication are at www.genesdev.org/cgi/doi/10.1101/gad.842500.

    • Received August 11, 2000.
    • Accepted September 19, 2000.
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