Abstract
The relationship between the DNA replication and spindle checkpoints of the cell cycle is unclear, given that in most eukaryotes, spindle formation occurs only after DNA replication is complete. Fission yeast rad3 mutant cells, which are deficient in DNA replication checkpoint function, enter, progress through, and exit mitosis even when DNA replication is blocked. In contrast, the entry of cds1 mutant cells into mitosis is delayed by several hours when DNA replication is inhibited. We show here that this delay in mitotic entry in cds1 cells is due in part to activation of the spindle checkpoint protein Mad2p. In the presence of the DNA replication inhibitor hydroxyurea (HU), cds1 mad2 cells entered and progressed through mitosis earlier than did cds1 cells. Overexpression of Mad2p or inactivation of Slp1p, a regulator of the anaphase-promoting complex, also rescued the checkpoint defect of HU-treated rad3 cells. Rad3p was shown to be involved in the physical interaction between Mad2p and Slp1p in the presence of HU. These results suggested that Mad2p and Slp1p act downstream of Rad3p in the DNA replication checkpoint and that Mad2p is required for the DNA replication checkpoint when Cds1p is compromised.
Highlights
Cell cycle checkpoints monitor cell cycle progression to ensure the integrity of the genome and the fidelity of sister chromatid separation [1,2,3]
Premature entry into mitosis causes cells to divide in a reduced cell size, whereas activation of the DNA replication checkpoint prevents or delays entry into mitosis, resulting in elongated cells
We tested whether the spindle checkpoint proteins Mad1, Mad2, Mad3, and Bub1 are required for the DNA replication checkpoint [19, 20]
Summary
Cell cycle checkpoints monitor cell cycle progression to ensure the integrity of the genome and the fidelity of sister chromatid separation [1,2,3]. We show here that this delay in mitotic entry in cds1 cells is due in part to activation of the spindle checkpoint protein Mad2p. Exposure to HU inhibited the completion of DNA replication by at least 8 h as judged by flow cytometric analysis (Fig. 1c) and resulted in a marked increase of elongated cells in wild-type (wt), whereas with HU treatment the rad3 mutant (rad3) cells were smaller than wt cells (Fig. 1, a and b), as reported previously [2, 3].
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