Involvement of Schizosaccharomyces pombe Srs2 in cellular responses to DNA damage

In the budding yeast Saccharomyces cerevisiae the Srs2/RadH DNA helicase promotes survival after ultraviolet (UV) irradiation, and has been implicated in DNA repair, recombination and checkpoint signalling following DNA damage. A second helicase, Sgs1, is the S.cerevisiae homologue of the human BLM and WRN proteins, which are defective in cancer predisposition and/or premature ageing syndromes. Saccharomyces cerevisiae cells lacking both Srs2 and Sgs1 exhibit a severe growth defect. We have identified an Srs2 orthologue in the fission yeast Schizosaccharomyces pombe, and have investigated its role in responses to UV irradiation and inhibition of DNA replication. Deletion of fission yeast srs2 caused spontaneous hyper-recombination and UV sensitivity, and simultaneous deletion of the SGS1 homologue rqh1 caused a severe growth defect reminiscent of that seen in the equivalent S.cerevisiae mutant. However, unlike in budding yeast, inactivation of the homologous recombination pathway did not suppress this growth defect. Indeed, the homologous recombination pathway was required for maintenance of normal fission yeast viability in the absence of Srs2, and loss of homologous recombination and loss of Srs2 contributed additively to UV sensitivity. We conclude that Srs2 plays related, but not identical, roles in the two yeast species.     

The role of novel genes rrp1+ and rrp2+ in the repair of DNA damage in Schizosaccharomyces pombe

We identified two predicted proteins in Schizosaccharomyces pombe, Rrp1 (SPAC17A2.12) and Rrp2 (SPBC23E6.02) that share 34% and 36% similarity to Saccharomyces cerevisiae Ris1p, respectively. Ris1p is a DNA-dependent ATP-ase involved in gene silencing and DNA repair. Rrp1 and Rrp2 also share similarity with S. cerevisiae Rad5 and S. pombe Rad8, containing SNF2-N, RING finger and Helicase-C domains. To investigate the function of the Rrp proteins, we studied the DNA damage sensitivities and genetic interactions of null mutants with known DNA repair mutants. Single Δrrp1 and Δrrp2 mutants were not sensitive to CPT, 4NQO, CDPP, MMS, HU, UV or IR. The double mutants Δrrp1 Δrhp51 and Δrrp2 Δrhp51 plus the triple Δrrp1 Δrrp2 Δrhp51 mutant did not display significant additional sensitivity. However, the double mutants Δrrp1 Δrhp57 and Δrrp2 Δrhp57 were significantly more sensitive to MMS, CPT, HU and IR than the Δrhp57 single mutant. The checkpoint response in these strains was functional. In S. pombe, Rhp55/57 acts in parallel with a second mediator complex, Swi5/Sfr1, to facilitate Rhp51-dependent DNA repair. Δrrp1 Δsfr1 and Δrrp2 Δsfr1 double mutants did not show significant additional sensitivity, suggesting a function for Rrp proteins in the Swi5/Sfr1 pathway of DSB repair. Consistent with this, Δrrp1 Δrhp57 and Δrrp2 Δrhp57 mutants, but not Δrrp1 Δsfr1 or Δrrp2 Δsfr1 double mutants, exhibited slow growth and aberrations in cell and nuclear morphology that are typical of Δrhp51.     

Schizosaccharomyces pombe Swi1, Swi3, and Hsk1 are components of a novel S-phase response pathway to alkylation damage

The Swi1 and Swi3 proteins are required for mat1 imprinting and mating-type switching in Schizosaccharomyces pombe, where they mediate a pause of leading-strand replication in response to a lagging-strand signal. In addition, Swi1 has been demonstrated to be involved in the checkpoint response to stalled replication forks, as was described for the Saccharomyces cerevisiae homologue Tof1. This study addresses the roles of Swi1 and Swi3 during a replication process perturbed by the presence of template bases alkylated by methyl methanesulfonate (MMS). Both the swi1 and swi3 mutations have additive effects on MMS sensitivity and on the MMS-induced damage checkpoint response when combined with chk1 and cds1, but they are nonadditive with hsk1. Cells with swi1, swi3, or hsk1 mutations are also defective in slowing progression through S phase in response to MMS damage. Moreover, swi1 and swi3 strains show increased levels of genomic instability even in the absence of exogenously induced DNA damage. Chromosome fragmentation, increased levels of single-stranded DNA, increased recombination, and instability of replication forks stalled in the presence of hydroxyurea are observed, consistent with the possibility that the replication process is affected in these mutants. In conclusion, Swi1, Swi3, and Hsk1 act in a novel S-phase checkpoint pathway that contributes to replication fork maintenance and to survival of alkylation damage.     

Schizosaccharomyces pombe Ddb1 is functionally linked to the replication checkpoint pathway

Schizosaccharomyces pombe Ddb1 is homologous to the mammalian DDB1 protein, which has been implicated in damaged-DNA recognition and global genomic repair. However, a recent study suggested that the S. pombe Ddb1 is involved in cell division and chromosomal segregation. Here, we provide evidence that the S. pombe Ddb1 is functionally linked to the replication checkpoint control gene cds1. We show that the S. pombe strain lacking ddb1 has slow growth due to delayed replication progression. Flow cytometric analysis shows an extensive heterogeneity in DNA content. Furthermore, the Deltaddb1 strain is hypersensitive to UV irradiation in S phase and is unable to tolerate a prolonged replication block imposed by hydroxyurea. Interestingly, the Deltaddb1 strain exhibits a high level of the Cds1 kinase activity during passage through S phase. Moreover, mutation of the cds1 gene relieves the defects observed in Deltaddb1 strain. The results suggest that many of the defects observed in Deltaddb1 cells are linked to an aberrant activation of Cds1, and that Ddb1 is functionally linked to Cds1.     

Redundant roles of Srs2 helicase and replication checkpoint in survival and rDNA maintenance in Schizosaccharomyces pombe

Srs2 helicase is believed to function as an anti-recombinase by resolving inappropriate Rad51-DNA filament. We found synthetic lethality or poor growth of srs2 with rad3 or mrc1 in Schizossacharomyces pombe. Lethality may result from a defect in non-checkpoint function of Rad3 or Mrc1 in the absence of Srs2, because srs2∆ rad9∆, srs2∆ chk1∆ cds1∆ or srs2∆ mrc1-14A (non-phosphorylatable mrc1 allele) did not show significant growth impairment. Notably, the inactivation of rhp51/RAD51 or rad22/RAD52 failed to rescue the growth, suggesting that events that impose lethality are independent of homologous recombination. Incubation of the conditional srs2∆ rad3 ^ts cells at restrictive temperature led not only to a viability decrease but also to a remarkable shortening of rDNA clusters (~100 copies). As opposed to the growth defect, shortening of rDNA clusters was also observed in srs2∆ rad9∆, srs2∆ chk1∆ cds1∆ or srs2∆ mrc1-14A, indicating that proper replication checkpoint signaling is critical for rDNA maintenance. Activation of Chk1 in the unchallenged mrc1-14A srs2∆ cells implies a certain level of spontaneous fork damage that might be the cause for rDNA instability. The data suggest that redundant functions of Srs2 and checkpoint proteins are essential for two independent aspects of genome maintenance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fission yeast Swi5/Sfr1 and Rhp55/Rhp57 differentially regulate Rhp51-dependent recombination outcomes

Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, we analyzed in vivo functions of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Delta mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Delta mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage-induced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5Delta and rhp57Delta mutants and completely abolished in an swi5Delta rhp57Delta double mutant. An assay for products generated by HO endonuclease-induced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator.     

DNA binding domain in the replication checkpoint protein Mrc1 of Schizosaccharomyces pombe

The replication checkpoint is activated when replication forks are obstructed by DNA lesions or protein complexes bound to DNA or when DNA synthesis is restrained by the limited availability of deoxyribonucleotides. This checkpoint preserves genome integrity by stabilizing stalled forks and delaying the onset of mitosis. In the fission yeast Schizosaccharomyces pombe, Mrc1 is a replication checkpoint adaptor protein that allows the sensor kinase Rad3-Rad26 to activate the effector kinase Cds1. In Saccharomyces cerevisiae, Mrc1 associates with replication forks and co-precipitates with the DNA replication protein Cdc45. Whether or not Mrc1 interacts directly with DNA is unknown. Here we define a approximately 150 amino acid DNA binding domain (DBD) in the N-terminal region of S. pombe Mrc1. The DBD interacts preferentially with branched DNA structures in vitro. Deletion of the DBD or point mutations that diminish its DNA binding activity render cells sensitive to the replication inhibitor hydroxyurea. These mutations also impair the replication checkpoint arrest. The DBD has a helix-loop-helix motif that is predicted to bind DNA. This motif is conserved in the recently identified N-terminal DBD of human Claspin, a presumptive homolog of yeast Mrc1 proteins.     

A novel replication arrest pathway in response to DNA damage

DNA damage has been shown to regulate DNA replication both by inhibition of origin utilization, and by slowing of replication progression. We have recently reported another mechanism by which DNA damage affects replication, in which the presence of damaged DNA inhibits, in trans, the initiation of chromosomal replication. This inhibition occurs by blocking the association of the processivity clamp PCNA with undamaged chromatin. This inhibitory activity is not due to sequestration of replication factors by the damaged DNA, rather, it acts through generation of a diffusible inhibitor of PCNA loading. The activation of this pathway is independent of canonical checkpoint signaling, and, in fact, results in activation of the checkpoint. This novel pathway may therefore represent an amplification step to stop cell cycle progression in response to lower levels of DNA damage.     

Schizosaccharomyces pombe Rad4/Cut5 protein modification and chromatin binding changes in DNA damage

The Schizosaccharomyces pombe Rad4/Cut5 protein is essential for DNA replication and checkpoint control. We have analyzed the behavior of the protein during unperturbed DNA replication, in different replication and checkpoint mutant backgrounds and in response to DNA-damaging agents. In an unperturbed cell cycle, Rad4 is chromatin bound and the mobility of the protein is not altered. Rad4 protein level and thus chromatin binding are dependent on a functional DNA polymerase epsilon. In response to replication arrest and DNA damage, the protein is modified in a Rad3-dependent manner. These data indicate that Rad4 undergoes diverse forms of regulation that are distinct in both DNA replication and checkpoint response.     

Pnk1, a DNA kinase/phosphatase required for normal response to DNA damage by gamma-radiation or camptothecin in Schizosaccharomyces pombe.

We report the characterization of Pnk1, a 45-kDa homolog of the human polynucleotide kinase PNKP in Schizosaccharomyces pombe. Recombinant Pnk1 like human PNKP exhibits both 5'-DNA kinase and 3'-DNA phosphatase activities in vitro. Furthermore, we detected 3'-DNA phosphatase activity with a single-stranded substrate in extracts from wild-type yeast, but no activity was detected in pnk1delta strains. We have shown that GFP-tagged Pnk1 like mammalian PNKP localizes to the nucleus. Deletion of pnk1 does not affect cell growth under normal conditions but results in significant hypersensitivity to gamma-radiation or camptothecin, an inhibitor of topoisomerase I, suggesting that Pnk1 plays an important role in the repair of DNA strand breaks produced by these agents. The pnk1 deletion mutants were not hypersensitive to ethyl methanesulfonate, methyl methanesulfonate, or 4-nitroquinoline N-oxide. Expression of human PNKP in pnk1delta cells restores resistance to gamma-radiation or camptothecin, suggesting that the functions of yeast Pnk1 and human PNKP have been conserved.     

Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity

In Saccharomyces cerevisiae the rate of DNA replication is slowed down in response to DNA damage as a result of checkpoint activation, which is mediated by the Mec1 and Rad53 protein kinases. We found that the Srs2 DNA helicase, which is involved in DNA repair and recombination, is phosphorylated in response to intra-S DNA damage in a checkpoint-dependent manner. DNA damage-induced Srs2 phosphorylation also requires the activity of the cyclin-dependent kinase Cdk1, suggesting that the checkpoint pathway might modulate Cdk1 activity in response to DNA damage. Moreover, srs2 mutants fail to activate Rad53 properly and to slow down DNA replication in response to intra-S DNA damage. The residual Rad53 activity observed in srs2 cells depends upon the checkpoint proteins Rad17 and Rad24. Moreover, DNA damage-induced lethality in rad17 mutants depends partially upon Srs2, suggesting that a functional Srs2 helicase causes accumulation of lethal events in a checkpoint-defective context. Altogether, our data implicate Srs2 in the Mec1 and Rad53 pathway and connect the checkpoint response to DNA repair and recombination.     

Sexual reproduction as a response to H2O2 damage in Schizosaccharomyces pombe.

Although sexual reproduction is widespread, its adaptive advantage over asexual reproduction is unclear. One major advantage of sex may be its promotion of recombinational repair of DNA damage during meiosis. This idea predicts that treatment of the asexual form of a facultatively sexual-asexual eucaryote with a DNA-damaging agent may cause it to enter the sexual cycle more frequently. Endogenous hydrogen peroxide is a major natural source of DNA damage. Thus, we treated vegetative cells of Schizosaccharomyces pombe with hydrogen peroxide to test if sexual reproduction increases. Among untreated stationary-phase S. pombe populations the sexual spores produced by meiosis represented about 1% of the total cells. However, treatment of late-exponential-phase vegetative cells with hydrogen peroxide increased the percentage of meiotic spores in the stationary phase by 4- to 18-fold. Oxidative damage therefore induces sexual reproduction in a facultatively sexual organism, a result expected by the hypothesis that sex promotes DNA repair.     

Swi2/Snf2-like protein Uls1 functions in the Sgs1-dependent pathway of maintenance of rDNA stability and alleviation of replication stress

The Saccharomyces cerevisiae Uls1 belongs to the Swi2/Snf2 family of DNA-dependent ATPases and a new protein family of SUMO-targeted ubiquitin ligases. Here we show that Uls1 is implicated in DNA repair independently of the replication stress response pathways mediated by the endonucleases Mus81 and Yen1 and the helicases Mph1 and Srs2. Uls1 works together with Sgs1 and we demonstrate that the attenuation of replication stress-related defects in sgs1Δ by deletion of ULS1 depends on a functional of Rad51 recombinase and post-replication repair pathway mediated by Rad18 and Rad5, but not on the translesion polymerase, Rev3. The higher resistance of sgs1Δ uls1Δ mutants to genotoxic stress compared to single sgs1Δ cells is not the result of decreased formation or accelerated resolution of recombination-dependent DNA structures. Instead, deletion of ULS1 restores stability of the rDNA region in sgs1Δ cells. Our data suggest that Uls1 may contribute to genomic stability during DNA synthesis and channel the repair of replication lesions into the Sgs1-dependent pathway, with DNA translocase and SUMO binding activities of Uls1 as well as a RING domain being essential for its functions in replication stress response.     

Schizosaccharomyces pombe sxa1+ and sxa2+ encode putative proteases involved in the mating response.

The Schizosaccharomyces pombe sxa1 and sxa2 mutants showed an exaggerated response to mating pheromones, producing excessively long conjugation tubes and exhibiting mating deficiency. This phenotype was similar to phenotypes of cells bearing an activated allele of ras1, such as ras1Val-17 or ras1Leu-66, and phenotypes of cells defective in gap1. However, genetic evidence suggested that the sxa1 and sxa2 gene products are not directly involved in the Ras1 pathway. The gene products of sxa1 and sxa2, as deduced from their nucleotide sequences, were homologous to aspartyl proteases and serine carboxypeptidases, respectively. The sxa1 gene function was required for efficient mating only in h+ cells, although even disruption of sxa1 did not completely abolish the mating ability. Conversely, the sxa2 gene function was required only in h- cells. Wild-type cells produced a diffusible substance, which may be the sxa2 gene product itself, that could confer fertility to sxa2 mutant cells placed at a distance. These observations are consistent with the possibility that the sxa gene products are involved in degradation or processing of the mating pheromones and that their loss cause a persistent response to the pheromones.     

Ddb1 is required for the proteolysis of the Schizosaccharomyces pombe replication inhibitor Spd1 during S phase and after DNA damage

Recently we showed that the Schizosaccharomyces pombe ddb1 gene plays a role in S phase progression. A mutant S. pombe strain lacking expression of the ddb1 gene exhibited slow replication through both early and late regions causing a slow S phase phenotype. We attributed the phenotypes in the ddb1 strain to an increased activity of the replication checkpoint kinase Cds1. However, the basis for a high basal Cds1 activity in the ddb1 strain was not clear. It was shown that Ddb1 associates with the Cop9/signalosome. Moreover, the phenotypes of the Deltaddb1 strain are remarkably similar to the Deltacsn1 (or Deltacsn2) strain that lacks expression of the Csn1 (or Csn2) subunit of the Cop9/signalosome. Cop9/signalosome cooperates with Pcu4 to induce proteolysis of Spd1, which inhibits DNA replication by inhibiting ribonucleotide reductase. Therefore, we investigated whether Ddb1 is required for the proteolysis of Spd1. Here we show that a S. pombe strain lacking expression of Ddb1 fails to induce proteolysis of Spd1 in S phase and after DNA damage. Moreover, deletion of the spd1 gene attenuates the Cds1 kinase activity in cells lacking the expression of ddb1, suggesting that an accumulation of Spd1 results in the increase of Cds1 activity in the Deltaddb1 strain. In addition, the double mutant lacking spd1 and ddb1 no longer exhibits the growth defects and DNA damage sensitivity observed in the Deltaddb1 strain. Our results establish an essential role of Ddb1 in the proteolysis of Spd1. In addition, the observation provides evidence for a functional link between Ddb1 and the Cop9/signalosome.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea.     

Characterisation of the Schizosaccharomyces pombe rad4/cut5 mutant phenotypes: dissection of DNA replication and G2 checkpoint control function

Mutation of the essential Schizosaccharomyces pombe rad4/cut5 gene causes sensitivity to UV and ionising radiation at the permissive temperature whilst at the restrictive temperature cells fail to undergo DNA replication but still attempt mitosis owing to a defective S-phase checkpoint response. Many mutations in genes encoding DNA replication proteins also abolish checkpoint responses, possibly because the replication machinery is a pre-requisite for the generation of the signal. We demonstrate here that rad4/cut5 cells fail to arrest cell division when treated with the replication inhibitor hydroxyurea at the semi-permissive temperature 32°?C, but retain essentially normal replicative capacity. This demonstrates that the replication and checkpoint function of the rad4/cut5 gene product can be separated and that the Rad4 protein differs from other replication proteins in being directly involved in generating the S-phase checkpoint signal. Furthermore, we have investigated the checkpoint response or rad4/cut5-deficient cells to γ-irradiation and UV-mimetic drugs. We find that, at the restrictive temperature, the rad4 ^? /cut5 ^? cells fail to delay mitosis in response to γ-irradiation whilst retaining a normal checkpoint response to the UV-mimetic drug 4-nitroquinoline-1-oxide. The lack of the γ-irradiation checkpoint is reminiscent of the deficiency associated with mutation of the human ATM locus, the causative deficiency of the heritable disorder ataxia telangiectasia. The implications of our results for the organisation of distinct checkpoint-response pathways in both fission yeast and mammalian cells are discussed. Moreover the data are consistent with a model in which the generation of the S-Phase checkpoint signal is DNA polymerase ? dependent.