Checkpoints that respond to DNA structure changes were originally defined by the inability of yeast mutants to prevent mitosis following DNA damage or S-phase arrest. Genetic analysis has subsequently identified subpathways of the DNA structure checkpoints, including the reversible arrest of DNA synthesis. Here, we show that the Cds1 kinase is required to slow S phase in the presence of DNA-damaging agents. Cds1 is phosphorylated and activated by S-phase arrest and activated by DNA damage during S phase, but not during G 1 or G 2 . Activation of Cds1 during S phase is dependent on all six checkpoint Rad proteins, and Cds1 interacts both genetically and physically with Rad26. Unlike its Saccharomyces cerevisiae counterpart Rad53, Cds1 is not required for the mitotic arrest checkpoints and, thus, defines an S-phase specific subpathway of the checkpoint response. We propose a model for the DNA structure checkpoints that offers a new perspective on the function of the DNA structure checkpoint proteins. This model suggests that an intrinsic mechanism linking S phase and mitosis may function independently of the known checkpoint proteins.[Key Words: Cds1 kinase; S-phase arrest; DNA structure checkpoints; S. pombe] Received August 11, 1997; revised version accepted November 24, 1997.Checkpoint pathways that respond to changes in DNA structure ensure the integrity of the DNA. After detection of specific DNA or DNA-protein structures, a signal is transduced to effector molecules that implement checkpoint-dependent responses such as cell-cycle arrest (Elledge 1996). Many components of the DNA-structure checkpoint pathways have been identified (Carr and Hoekstra 1995). In the fission yeast Schizosaccharomyces pombe, a group of six checkpoint Rad proteins (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1) are thought to participate in the monitoring and signaling processes that detect both DNA damage and incomplete DNA replication (Al-Khodairy and Carr 1992;Enoch et al. 1992;Rowley et al. 1992;Al-Khodairy et al. 1994). Central to this group is the Rad3 protein, which shares homology with both budding yeast and human checkpoint proteins (Savitsky et al. 1995;Bentley et al. 1996;Cimprich et al. 1996). Rad3 is a member of a larger subfamily of protein kinases that share structural similarities. This subfamily consists of large proteins with a lipid kinase-related domain at the carboxyl terminus. One member, DNA-PKcs, is well characterized as a protein kinase that is activated by association with DNA-binding subunits (Jeggo et al. 1995). By analogy with DNA-PK, we have proposed that Rad3 is activated by the other checkpoint Rad proteins, which may interact with the specific DNA or DNA-protein structures generated by DNA damage and DNA synthesis (Carr 1997).DNA structure checkpoints respond to several distinct signals. The best characterized are DNA damage caused by UV or ␥-irradiation and S-phase arrest resulting from hydroxyurea (HU) exposure. In response to DNA damage, but not S-phase arrest, Chk1 kinase becomes phosphorylated in a m...

No abstract

The dependence of cell-cycle progression on the integrity of the genome has been described as checkpoint control. A number of mutants of the fission yeast Schizosaccharomyces pombe, selected for their sensitivity to DNA damage caused by radiation (rad mutants) or to the DNA synthesis inhibitor hydroxyurea (hus mutants) have been classified as checkpoint mutants because they fail to arrest the cell cycle in response to DNA damage or incompletely replicated DNA. Coupling of the checkpoint pathways that monitor DNA repair and replication to control of the cell cycle is essential. In a search for components that interact with the cell-cycle regulatory kinase p34cdc2, we have identified a novel fission yeast protein kinase homologue which is involved in cell-cycle arrest when DNA damage has occurred or when unligated DNA is present. We have called the gene encoding this protein chk1 for checkpoint kinase. Multiple copies of chk1 partially rescue the ultraviolet sensitivity of rad1-1, a mutant deficient in checkpoint control. Identification of a gene involved in checkpoint control as a rescue of a cdc2 mutant links the rad1-dependent DNA-damage-sensing pathway and p34cdc2 activity.

Exposure of eukaryotic cells to agents that generate DNA damage results in transient arrest of progression through the cell cycle. In fission yeast, the DNA damage checkpoint associated with cell cycle arrest before mitosis requires the protein kinase p56chk1. DNA damage induced by ultraviolet light, gamma radiation, or a DNA-alkylating agent has now been shown to result in phosphorylation of p56chk1. This phosphorylation decreased the mobility of p56chk1 on SDS-polyacrylamide gel electrophoresis and was abolished by a mutation in the p56chk1 catalytic domain, suggesting that it might represent autophosphorylation. Phosphorylation of p56chk1 did not occur when other checkpoint genes were inactive. Thus, p56chk1 appears to function downstream of several of the known Schizosaccharomyces pombe checkpoint gene products, including that encoded by rad3+, a gene with sequence similarity to the ATM gene mutated in patients with ataxia telangiectasia. The phosphorylation of p56chk1 provides an assayable biochemical response to activation of the DNA damage checkpoint in the G2 phase of the cell cycle.

No abstract

No abstract

Mutant alleles of SEC4, an essential gene required for the final stage of secretion in yeast, have been generated by in vitro mutagenesis. Deletion of the two cysteine residues at the C terminus of the protein results in a soluble non‐functional protein, indicating that those two residues are required for normal localization of Sec4p to secretory vesicles and the plasma membrane. A mutant allele of SEC4 generated to mimic an activated, transforming allele of H‐ras, as predicted, does not bind GTP. The presence of this allele in cells containing wild‐type SEC4 causes a secretory defect and the accumulation of secretory vesicles. The results of genetic studies indicate that this allele behaves as a dominant loss of function mutant and as such prevents wild‐type protein from functioning properly. We propose a model in which Sec4p cycles between an active and an inactive state in order to mediate the fusion of vesicles to the plasma membrane.

In the fission yeast Schizosaccharomyces pombe, the protein kinase Chk1 has an essential role in transducing a delay signal to the cell cycle machinery in the presence of DNA damage. Fission yeast cells lacking the chk1 gene do not delay progression of the cell cycle in response to damage and are thus sensitive to DNA damaging agents. We have previously shown that Chk1 is phosphorylated following DNA damage induced by a variety of agents and that this is dependent on the integrity of the DNA damage checkpoint pathway, including Rad3, the ATR homolog. Through a combination of mutagenesis and phospho-specific antibodies, we have shown that serine at position 345 (S345) is phosphorylated in vivo in response to DNA damage, and that S345 phosphorylation is required for an intact checkpoint response. We have developed a kinase assay for Chk1, and have shown that basal Chk1 kinase activity is increased in response to DNA damage and that this increase, but not the basal activity, is dependent on S345. Furthermore, we show that S345 phosphorylation is required for Chk1 to associate with Rad24, a 14-3-3 protein, upon DNA damage. These results are consistent with a model whereby Chk1 phosphorylation results in increased Chk1 kinase activity that is necessary for both checkpoint delay and cellular survival following damage to the genome. These data are similar to observations made in mammalian cells and Xenopus oocyte extracts, suggesting that mechanisms leading to Chk1 activation have been conserved in evolution.