Mus81-Eme1-dependent and -independent crossovers form in mitotic cells during double-strand break repair in Schizosaccharomyces pombe.

During meiosis, double-strand breaks (DSBs) lead to crossovers, thought to arise from the resolution of double Holliday junctions (HJs) by an HJ resolvase. In Schizosaccharomyces pombe, meiotic crossovers are produced primarily through a mechanism requiring the Mus81-Eme1 endonuclease complex. Less is known about the processes that produces crossovers during the repair of DSBs in mitotic cells. We employed an inducible DSB system to determine the role of Rqh1-Top3 and Mus81-Eme1 in mitotic DSB repair and crossover formation in S. pombe. In agreement with the meiotic data, crossovers are suppressed in cells lacking Mus81-Eme1. And relative to the wild type, rqh1Delta cells show a fourfold increase in crossover frequency. This suppression of crossover formation by Rqh1 is dependent on its helicase activity. We found that the synthetic lethality of cells lacking both Rqh1 and Eme1 is suppressed by loss of swi5(+), which allowed us to show that the excess crossovers formed in an rqh1Delta background are independent of Mus81-Eme1. This result suggests that a second process for crossover formation exists in S. pombe and is consistent with our finding that deletion of swi5(+) restored meiotic crossovers in eme1Delta cells. Evidence suggesting that Rqh1 also acts downstream of Swi5 in crossover formation was uncovered in these studies. Our results suggest that during Rhp51-dependent repair of DSBs, Rqh1-Top3 suppresses crossovers in the Rhp57-dependent pathway while Mus81-Eme1 and possibly Rqh1 promote crossovers in the Swi5-dependent pathway.

A postsynaptic role for Rhp55/57 that is responsible for cell death in Deltarqh1 mutants following replication arrest in Schizosaccharomyces pombe.

Following replication arrest, multiple cellular responses are triggered to maintain genomic integrity. In fission yeast, the RecQ helicase, Rqh1, plays a critical role in this process. This is demonstrated in Deltarqh1 cells that, following treatment with hydroxyurea (HU), undergo an aberrant mitosis leading to cell death. Previous data suggest that Rqh1 functions with homologous recombination (HR) in recovery from replication arrest. We have found that loss of the HR genes rhp55(+) or rhp57(+), but not rhp51(+) or rhp54(+), suppresses the HU sensitivity of Deltarqh1 cells. Much of this suppression requires Rhp51 and Rhp54. In addition, this suppression is partially dependent on swi5(+). In budding yeast, overexpressing Rad51 (the Rhp51 homolog) minimized the need for Rad55/57 (Rhp55/57) in nucleoprotein filament formation. We overexpressed Rhp51 in Schizosaccharomyces pombe and found that it greatly reduced the requirement for Rhp55/57 in recovery from DNA damage. However, overexpressing Rhp51 did not change the Deltarhp55 suppression of the HU sensitivity of Deltarqh1, supporting an Rhp55/57 function during HR independent of nucleoprotein filament formation. These results are consistent with Rqh1 playing a role late in HR following replication arrest and provide evidence for a postsynaptic function for Rhp55/57.

The severe slow growth of Deltasrs2 Deltarqh1 in Schizosaccharomyces pombe is suppressed by loss of recombination and checkpoint genes.

Our interest in the Schizosaccharomyces pombe RecQ helicase, rqh1+, led us to investigate the function of a related putative DNA helicase, srs2+. We identified the srs2+ homolog in S.pombe, and found that srs2+ is not essential for cell viability. A Deltasrs2 Deltarqh1 double mutant grows extremely slowly with aberrant shaped cells and low viability. This slow growth does not appear to be related to stalled replication, as Deltasrs2 Deltarqh1 cells showed higher survival rates, compared with Deltarqh1, when stalled forks were increased by UV irradiation or hydroxy urea treatment. Consistent with this result, we found that Deltasrs2 Deltarqh1 cells progress through S-phase with a slight delay, but undergo a checkpoint-dependent arrest presumably at G2/M. Further, we found that Deltasrs2 Deltarqh1 slow growth is related to recombination, as loss of either the rhp51+ or rhp57+ recombination genes improves cell growth in the double mutant. Deltasrs2 is also synthetic lethal with Deltarhp54, another homologous recombination gene. This lethality is suppressed in a Deltarhp51 background. Together, these results demonstrate a clear genetic interaction between rqh1+, srs2+ and the genes of the homologous recombination pathway.

A single unbranched S-phase DNA damage and replication fork blockage checkpoint pathway.

The eukaryotic intra-S-phase checkpoint, which slows DNA synthesis in response to DNA damage, is poorly understood. Is DNA damage recognized directly, or indirectly through its effects on replication forks? Is the slowing of S phase in part because of competition between DNA synthesis and recombination/repair processes? The results of our genetic analyses of the intra-S-phase checkpoint in the fission yeast, Schizosaccharomyces pombe, suggest that the slowing of S phase depends weakly on the helicases Rqh1 and Srs2 but not on other recombination/repair pathways. The slowing of S phase depends strongly on the six checkpoint-Rad proteins, on Cds1, and on Rad4/Cut5 (similar to budding yeast Dpb11, which interacts with DNA polymerase epsilon) but not on Rhp9 (similar to budding yeast Rad9, necessary for direct damage recognition). These results suggest that, in fission yeast, the signal activating the intra-S-phase checkpoint is generated only when replication forks encounter DNA damage.