Mechanism of Caffeine-Induced Checkpoint Override in Fission Yeast

Abstract

Mitotic checkpoints restrain the onset of mitosis (M) when DNA is incompletely replicated or damaged. These checkpoints are conserved between the fission yeast Schizosaccharomyces pombe and mammals. In both types of organisms, the methylxanthine caffeine overrides the synthesis (S)-M checkpoint that couples mitosis to completion of DNA S phase. The molecular target of caffeine was sought in fission yeast. Caffeine prevented activation of Cds1 and phosphorylation of Chk1, two protein kinases that enforce the S-M checkpoint triggered by hydroxyurea. Caffeine did not inhibit these kinases in vitro but did inhibit Rad3, a kinase that regulates Cds1 and Chk1. In accordance with this finding, caffeine also overrode the G₂-M DNA damage checkpoint that requires Rad3 function. Rad3 coprecipitated with Cds1 expressed at endogenous amounts, a finding that supports the hypothesis that Rad3 is involved in direct activation of Cds1.

ACKNOWLEDGMENTS

The first two authors contributed equally to this work.

Michael N. Boddy and Nick Rhind made helpful comments and suggestions; Antony Carr supplied strains; Alessandra Blasina, Takashi Toda, Chris Norbury, and Clare McGowan discussed results prior to publication. Members of the Scripps Cell Cycle Groups provided support and encouragement.

J.M.B. was supported by INSERM (France). B.A.M. was supported by the Schweizer Krebsliga and the Deutsche Forshungsgemeinschaft. This work was funded by NIH.

ADDENDUM IN PROOF

Since submission of this paper, there have appeared three additional reports of caffeine inhibiting the Rad3-related kinase ATM or ATR: C. A. Hall-Jackson et al. (Oncogene 18:6707–6713, 1999), J. N. Sarkaria et al. (Cancer Res. 59:4375–4382, 1999), and B. B. Zhou et al. (J. Biol. Chem.275:10342–10348, 2000).