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...

A system is described for gene disruption and replacement in Schizosaccharomyces pombe based on the homologous selectable marker, ura4, the structural gene for orotidine-5'-phosphate decarboxylase. The presence of a single copy of the wild-type gene can rescue a ura4 auxotrophic mutant. Furthermore, ura4- cells can be selected for in the presence of 5-fluoroorotic acid (5-FOA). This allows a convenient means of selecting for both forward and backward mutations. The sequence of a 1.8 kb HindIII fragment which contains the functional gene is reported. It encodes a single open reading frame of 264 amino acids which shows considerable conservation with the orotidine-5'-phosphate (OMP) decarboxylases from other organisms. The ura4 transcript is approximately 850 nucleotides long. It begins 51 bp upstream of the protein coding sequence and is unusual in that transcription termination occurs at or very close to the translational stop codon. To facilitate the use of ura4 in gene disruption experiments we have also constructed a novel strain of S. pombe called ura4-D18, in which the 1.8 kb HindIII fragment has been deleted from the chromosome. Using a combination of this strain and vectors containing ura4 as a selectable marker, we present a general method for targeting recombination events to the chromosomal locus under investigation.

The rad18 mutant of Schizosaccharomyces pombe is very sensitive to killing by both UV and ␥ radiation. We have cloned and sequenced the rad18 gene and isolated and sequenced its homolog from Saccharomyces cerevisiae, designated RHC18. The predicted Rad18 protein has all the structural properties characteristic of the SMC family of proteins, suggesting a motor function-the first implicated in DNA repair. Gene deletion shows that both rad18 and RHC18 are essential for proliferation. Genetic and biochemical analyses suggest that the product of the rad18 gene acts in a DNA repair pathway for removal of UV-induced DNA damage that is distinct from classical nucleotide excision repair. This second repair pathway involves the products of the rhp51 gene (the homolog of the RAD51 gene of S. cerevisiae) and the rad2 gene.Cells of all organisms have evolved an intricate series of DNA repair pathways to counteract the deleterious effects of all types of DNA damage. In Escherichia coli, nucleotide excision repair (NER) of UV damage requires the products of six genes. A complex of the UvrA and UvrB proteins binds to DNA and translocates to the site of the damage. The UvrC product then attaches to the complex, displacing UvrA, and the damaged DNA strand is nicked on both sides of the damaged site. The helicase activity of the UvrD product releases the oligonucleotide containing the damage, and DNA polymerase I and ligase complete the repair process (24). In eukaryotes, NER requires considerably more gene products, most of which are highly conserved (20). In Saccharomyces cerevisiae, the products of the RAD1, -2, -3, -4, -10, -14, and -25 genes are absolutely required for excision repair of UV damage, whereas there is only a partial requirement for RAD7, -16, and -23. There is evidence that the products of many of these genes form a multisubunit complex (e.g., see reference 53). In Schizosaccharomyces pombe, genes encoding highly homologous proteins have been identified (9,10,30,39), demonstrating the conservation of the classical NER pathway in this yeast.Null mutations in the S. cerevisiae NER genes RAD1, -2, -3, -10, and -14 result in a total deficiency in excision repair of UV-induced cyclobutane dimers and 6-4 photoproducts (28). Null mutants of the S. pombe homologs of RAD1 and RAD2 (rad16 and rad13, respectively), while showing many properties expected of excision repair-deficient mutants, are still able to excise UV-induced cyclobutane dimers and 6-4 photoproducts at a significant rate (7, 27). These results suggest that, in contrast to S. cerevisiae, there is a second pathway in S. pombe for removal of UV photoproducts.Most of the rad mutants of S. pombe are sensitive to both UV and ␥ irradiation. Some of these are involved in checkpoint control of the cell cycle to radiation (2, 3, 41). Others, which are particularly sensitive to ionizing radiation, are deficient in recombination repair (6,29,33,54). No mutant which is sensitive to ionizing but not to UV irradiation has yet been identified. The S. pombe rad18-X mutant ...

Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.

Gene amplification plays important roles in the progression of cancer and contributes to acquired drug resistance during treatment. Amplification can initiate via dicentric palindromic chromosome production and subsequent breakage-fusion-bridge cycles. Here we show that, in fission yeast, acentric and dicentric palindromic chromosomes form by homologous recombination protein-dependent fusion of nearby inverted repeats, and that these fusions occur frequently when replication forks arrest within the inverted repeats. Genetic and molecular analyses suggest that these acentric and dicentric palindromic chromosomes arise not by previously described mechanisms, but by a replication template exchange mechanism that does not involve a DNA double-strand break. We thus propose an alternative mechanism for the generation of palindromic chromosomes dependent on replication fork arrest at closely spaced inverted repeats.[Keywords: RTS7; template switch; recombination; gene amplification; breakage-fusion-bridge] Supplemental material is available at http://www.genesdev.org.