Schizosaccharomyces pombe Hsk1p Is a Potential Cds1p Target Required for Genome Integrity

Abstract

The fission yeast Hsk1p kinase is an essential activator of DNA replication. Here we report the isolation and characterization of a novel mutant allele of the gene. Consistent with its role in the initiation of DNA synthesis, hsk1^ts genetically interacts with several S-phase mutants. At the restrictive temperature,hsk1^ts cells suffer abnormal S phase and loss of nuclear integrity and are sensitive to both DNA-damaging agents and replication arrest. Interestingly, hsk1^tsmutants released to the restrictive temperature after early S-phase arrest in hydroxyurea (HU) are able to complete bulk DNA synthesis but they nevertheless undergo an abnormal mitosis. These findings indicate a second role for hsk1 subsequent to HU arrest. Consistent with a later S-phase role, hsk1^ts is synthetically lethal with Δrqh1 (RecQ helicase) orrad21ts (cohesin) mutants and suppressed by Δcds1 (RAD53 kinase) mutants. We demonstrate that Hsk1p undergoes Cds1p-dependent phosphorylation in response to HU and that it is a direct substrate of purified Cds1p kinase in vitro. These results indicate that the Hsk1p kinase is a potential target of Cds1p regulation and that its activity is required after replication initiation for normal mitosis.

ACKNOWLEDGMENTS

We are grateful to Hisao Masai for strains and plasmids and discussion of unpublished work. We thank Tamar Enoch, Greg Freyer, Hisao Ikeda, Paul Russell, Shelley Sazer, Nancy Walworth, and Mitsuhiro Yanagida for strains and plasmids; Sally Pasion and Dan Sherman for plasmids; Tony Hunter for antibodies; and Jeff Hodson, John Marlett, and Andrew Waight for excellent technical assistance. We are also much indebted to Magdalena Bezanilla, Mike Catlett, Eliana Gomez, Joel Leverson, Debbie Liang, and Sally Pasion for their critical readings of the manuscript.

This work was supported by American Cancer Society grant RPG-00-132-01-CCG (S.L.F.) and Medical Research Council of Canada grant MOP-36360 (G.W.B.). H.A.S. was partly supported by The Salk Institute President's Club and the Ralph M. Parsons Foundation. We acknowledge the Dreyfus Foundation for their support. S.L.F. is a scholar of the Leukemia and Lymphoma Society. G.W.B. is a special fellow of the Leukemia and Lymphoma Society.