Snaith H A, Brown G W, Forsburg S L
Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037-1099, USA.
Mol Cell Biol. 2000 Nov;20(21):7922-32. doi: 10.1128/MCB.20.21.7922-7932.2000.
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(ts) mutants 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 Deltarqh1 (RecQ helicase) or rad21ts (cohesin) mutants and suppressed by Deltacds1 (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.
裂殖酵母Hsk1p激酶是DNA复制的必需激活因子。在此，我们报告该基因一个新突变等位基因的分离与特性。与其在DNA合成起始中的作用一致，hsk1(ts)与多个S期突变体发生遗传相互作用。在限制温度下，hsk1(ts)细胞经历异常S期并丧失核完整性，且对DNA损伤剂和复制停滞均敏感。有趣的是，在羟基脲（HU）中早期S期停滞后释放到限制温度的hsk1(ts)突变体能够完成大量DNA合成，但它们仍会经历异常有丝分裂。这些发现表明hsk1在HU停滞之后具有第二个作用。与后期S期作用一致，hsk1(ts)与Deltarqh1（RecQ解旋酶）或rad21ts（黏连蛋白）突变体发生合成致死，并被Deltacds1（RAD53激酶）突变体抑制。我们证明Hsk1p在响应HU时发生Cds1p依赖性磷酸化，并且它在体外是纯化的Cds1p激酶的直接底物。这些结果表明Hsk1p激酶是Cds1p调控的潜在靶点，并且其活性在复制起始后对于正常有丝分裂是必需的。
