Isolation of DNA Structure-Dependent Checkpoint Mutants in S. pombe

Eukaryotic cells have the ability to influence progression through the cell cycle in response to internal and external inputs of “information”. They do so by using feedback control mechanisms able to arrest mitosis in response to different cellular events. Such active mechanisms capable of influencing the timing of cell-cycle events have been called “checkpoints” (1 ,2 ). Cells arrest progression through the cell cycle if they fail to complete DNA replication or if their DNA is damaged. The S-phase/mitosis (S-M) checkpoint plays a key role in the maintenance of the interdependency between S-phase and mitosis. Wild-type Schizosaccharomyces pombe (fission yeast) cells arrest cell-cycle progression in response to a DNA replication block, such as that induced by hydroxyurea (HU), but continue to grow in size, since they are still metabolically active. These cells are observed to have an elongated phenotype. Mutants have been isolated in S. pombe that have lost the S-M checkpoint and do not prevent mitosis if DNA replication during the previous S-phase is incomplete (3 –7 ). S-M checkpoint mutants do not delay cell-cycle events after exposure to HU, and will enter mitosis with unreplicated DNA. As a consequence, the elongated phenotype seen for wild-type cells is absent in checkpoint mutants. Instead, these mutants show a characteristic “cut” phenotype, where a cell has entered an abortive mitotic event followed by the formation of a septum through the nucleus. In these small dead cells, the nucleus is frequently cut in two by the septum and/or spread unevenly between both daughter cells.