Caffeine stabilizesCdc25 independently ofRad3 inSchizosaccharomyces pombecontributing to checkpoint override

Summary Cdc 25 is required for Cdc 2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc 25 and Cdc 2 in a Rad 3‐dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad 3‐related proteins in both S chizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc 25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc 25 accumulation in S . pombe independently of Rad 3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA ‐damaging agents. In the absence of Cdc 25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc 25 accumulation in S . pombe by suppressing its degradation independently of Rad 3. The induction of Cdc 25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine‐induced Cdc 25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc 25 accumulation on cell cycle progression is attenuated by Srk 1 and Mad 2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc 25.