Mechanism of caffeine-induced checkpoint override in fission yeast

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 G(2)-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.