DNA damage activates a cell-cycle checkpoint that prevents mitosis while DNA repair is under way. The protein Chk1 enforces this checkpoint by phosphorylating the mitotic inducer Cdc25. Phosphorylation of Cdc25 by Chk1 creates a binding site in Cdc25 for 14-3-3 proteins, but it is not known how 14-3-3 proteins regulate Cdc25. Rad24 is a 14-3-3 protein that is important in the DNA-damage checkpoint in fission yeast. Here we show that Rad24 controls the intracellular distribution of Cdc25. Elimination of Rad24 causes nuclear accumulation of Cdc25. Activation of the DNA-damage checkpoint causes the net nuclear export of Cdc25 by a process that requires Chk1, Rad24 and nuclear-export machinery. Mutation of a putative nuclear-export signal in Rad24 impairs the nuclear exclusion of Rad24, the damage-induced nuclear export of Cdc25 and the damage checkpoint. Thus, Rad24 appears to function as an attachable nuclear-export signal that enhances the nuclear export of Cdc25 in response to DNA damage.

Arrest of the cell cycle at the G2 checkpoint, induced by DNA damage, requires inhibitory phosphorylation of the kinase Cdc2 in both fission yeast and human cells. The kinase Wee1 and the phosphatase Cdc25, which regulate Cdc2 phosphorylation, were evaluated as targets of Chk1, a kinase essential for the checkpoint. Fission yeast cdc2-3w Deltacdc25 cells, which express activated Cdc2 and lack Cdc25, were responsive to Wee1 but insensitive to Chk1 and irradiation. Expression of large amounts of Chk1 produced the same phenotype as did loss of the cdc25 gene in cdc2-3w cells. Cdc25 associated with Chk1 in vivo and was phosphorylated when copurified in Chk1 complexes. These findings identify Cdc25, but not Wee1, as a target of the DNA damage checkpoint.

Cdc2, the kinase that induces mitosis, is regulated by checkpoints that couple mitosis to the completion of DNA replication and repair. The repair checkpoint kinase Chk1 regulates Cdc25, a phosphatase that activates Cdc2. Effectors of the replication checkpoint evoked by hydroxyurea (HU) are unknown. Treatment of fission yeast with HU stimulated the kinase Cds1, which appears to phosphorylate the kinase Wee1, an inhibitor of Cdc2. The protein kinase Cds1 was also required for a large HU-induced increase in the amount of Mik1, a second inhibitor of Cdc2. HU-induced arrest of cell division was abolished in cds1 chk1 cells. Thus, Cds1 and Chk1 appear to jointly enforce the replication checkpoint.

No abstract

A common cellular response to DNA damage is cell cycle arrest. This checkpoint control has been the subject of intensive genetic investigation, but the biochemical mechanism that prevents mitosis following DNA damage is unknown. In Schizosaccbaromyces potnbe, as well as vertebrates, the timing of mitosis under normal circumstances is determined by the balance of kinases and phosphatases that regulate inhibitory phosphorylation of Cdc2. In S. pombe, the phosphorylation occurs on tyrosine-15. This method of mitotic control is also used in S. pombe to couple mitosis with completion of DNA replication, but the role of Cdc2 tyrosine phosphorylation in the Chkl kinase-mediated DNA damage checkpoint has remained uncertain. We show that, in contrast to recent speculation, the Gj DNA damage checkpoint arrest in S. pombe depends on the inhibitory tyrosine phosphorylation of Cdc2 carried out by the Weel and Mikl kinases. Furthermore, the rate of Cdc2 tyrosine dephosphorylation is reduced by irradiation. This result implicates regulation of Cdc2 tyrosine dephosphorylation, mainly carried out by the Cdc25 tyrosine phosphatase, as an important part of the mechanism by which the DNA damage checkpoint induces Cdc2 inhibition and G2 arrest.