p56chk1 protein kinase is required for the DNA replication checkpoint at 37C in fission yeast

the timing of mitosis is determined by a gradual change Stefania Francesconi1, Muriel Grenon, in the wee1/cdc25 ratio. This ratio influences the cell size Dominique Bouvier and Giuseppe Baldacci at division and maintains the dependence of mitosis on IFC 1, Institut de Recherche sur le Cancer, CNRS UPR 9044, the completion of DNA replication (Enoch and Nurse, 7 rue Guy Moquet BP 8, 94801 Villejuif, France 1990; Moreno et al., 1990). Recently, it has been shown 1Corresponding author that during the S phase cell cycle block at the restrictive temperature of the thermosensitive cdc22-M45 mutant, Fission yeast p56chk1 kinase is known to be involved in the cdc25 phosphatase accumulates to high levels although the DNA damage checkpoint but not to be required its basal activity remains low. This indicates that the DNA for cell cycle arrest following exposure to the DNA replication checkpoint operates despite the accumulation replication inhibitor hydroxyurea (HU). For this of cdc25 protein (Kovelman and Russell, 1996). reason, p56chk1 is considered not to be necessary for Fission yeast mutants deficient in the DNA replication the DNA replication checkpoint which acts through the checkpoint have been isolated which fail to arrest the cell inhibitory phosphorylation of p34cdc2 kinase activity. In cycle in the presence of the DNA replication inhibitor a search for Schizosaccharomyces pombe mutants that hydroxyurea (HU) and thereby undergo mitotic catastrophe abolish the S phase cell cycle arrest of a thermosensitive (hus mutants). Analysis of hus mutants provided evidence DNA polymerase δ strain at 37°C, we isolated two for a partial overlap between DNA replication and DNA chk1 alleles. These alleles are proficient for the DNA damage checkpoints since many of these mutants were damage checkpoint, but induce mitotic catastrophe in also affected in the ability to arrest the cell cycle in G2 several S phase thermosensitive mutants. We show that phase after DNA damage, resulting in the radiationthe mitotic catastrophe correlates with a decreased sensitive phenotype (rad mutants). However, in fission level of tyrosine phosphorylation of p34cdc2. In addition, yeast, the radiation checkpoint seems not to involve we found that the deletion of chk1 and the chk1 the Y15 phosphorylation of p34cdc2. Indeed, mutations alleles abolish the cell cycle arrest and induce mitotic affecting elements involved in Y15 regulation do not catastrophe in cells exposed to HU, if the cells are abolish the G2 delay when cells are irradiated (Al-Khodairy grown at 37°C. These findings suggest that chk1 is and Carr, 1992; Enoch et al., 1992; Barbet and Carr, 1993; important for the maintenance of the DNA replication Sheldrick and Carr, 1993; Al-Khodairy et al., 1994). checkpoint in S phase thermosensitive mutants and The fission yeast checkpoint gene chk1 /rad27 has that the p56chk1 kinase must possess a novel function been proposed to be solely involved in the DNA damage that prevents premature activation of p34cdc2 kinase checkpoint, as deletion of this gene abolishes cell cycle under conditions of impaired DNA replication at 37°C. arrest after irradiation (rad phenotype) but does not abolish