Caffeine-mediated override of checkpoint controls: A requirement for rhp6 (Schizosaccharomyces pombe)

Cells exposed to inhibitors of DNA synthesis or suffering DNA damage are ar rested or delayed in interphase through the action of checkpoint controls. If the arrested cell is exposed to caffeine, relatively normal cell cycle p rogression is resumed and, as observed in checkpoint control mutants, loss of checkpoint control activity is associated with a reduction in cell viabi lity. To address the mechanism of caffeine's action on cell progression, fi ssion yeast mutants that take up caffeine but are not sensitized to hydroxy urea (HU) by caffeine were selected. Mutants 788 and 1176 are point mutants of rhp6, the fission yeast homolog of the budding yeast RAD6 gene. Mutant rhp6-788 is slightly HU sensitive, radiosensitive, and exhibits normal chec kpoint responses to HU, radiation, or inactivation of DNA ligase. However, the addition of caffeine does not override the associated cell cycle blocks . Both point and deletion mutations show synthetic lethality at room temper ature with temperature-sensitive mutations in cyclin B (cdc13-117) or the p hosphatase cdc25 (cdc25-22). These observations suggest that the rhp6 gene product, a ubiquitin-conjugating enzyme required for DNA damage repair, pro motes entry to mitosis in response to caffeine treatment.