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Regulation of the S-phase CDK activity required for the DNA replication by Nik1 and Swe1 kinases in S.cerevisiae

酿酒酵母中 Nik1 和 Swe1 激酶对 DNA 复制所需的 S 期 CDK 活性的调节

基本信息

批准号：
11680698
负责人：
NOJIMA Hiroshi
金额：
$ 2.37万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

In this research program, we have searched for novel cell cycle regulators in the budding yeast S.cerevisiae by cloning multicopy suppressors of the cdc2-L7 mutation of the fission yeast, S.pombe. One of the isolated genes encodes a putative protein kinase similar to Nim1 of S.pombe and was termed NIK1 (Nim1-like kinase 1). NIK1 is identical to HSL1, and the results obtained so far suggests that NIK1 is a nim1+homolog of S.cerevisiae. NIK1 has unique features as follows that have not been reported on nim1+ ; (1) the transcription of NIK1 was periodic with peaked at the G1/S boundary. (2) Ejnik1 cells showed HU and MMS sensitivity. (3) DNA was aberrantly distributed in the Ejnik1cdc28 double mutant. (4) Nik1 interacts with the Cdc28 complex genetically and biochemically. (5) NIK1 seemed to have genetic interaction with CDC6. These results suggests that Nik1 interacts with the Cdc28 complex and functions not only at the G2/M transition but also at S phase of the cell cycle. We analyzed on the phenotypes of nik1 Ej which were considered to be caused by some defects in DNA replication, i.e. HU sensitivity, higher plasmid instability and increased heat sensitivity in S-phase related ts mutant strains. These phenotypes were suppressed by swe1 Ej and Nik1 can phosphorylate Swe1, therefore, it was prospected that they were caused by the lowered activity of S phase Cdc28. Indeed, in vitro analysis of FLAG-tagged Clb5-dependent kinase activity revealed that the kinase activity of htis was inhibited by Swe1 as in the case of Clb2-dependent kinase. Moreover, cdc28Y19E mutant showed delayed initiation and progression of the DNA replication. Tyrosine-phosphorylated Cdc28 appears at S phase. These data indicate S-phase CDK complexes, at least including the Clb5-Cdc28 complex, is regulated by phosphorylation of conserved Tyrosine.

在本研究项目中，我们通过克隆裂变酵母S.pombe的cdc2-L7突变的多拷贝抑制子，在芽殖酵母s.c reevisiae中寻找新的细胞周期调节因子。其中一个分离的基因编码一种推测类似于S.pombe Nim1的蛋白激酶，并被命名为NIK1 （Nim1样激酶1）。NIK1与HSL1相同，目前得到的结果表明NIK1是S.cerevisiae的nim1+同源物。NIK1具有以下nim1+没有报道的独特功能；(1) NIK1的转录具有周期性，在G1/S边界达到峰值。(2) Ejnik1细胞具有HU和MMS敏感性。(3) Ejnik1cdc28双突变体DNA分布异常。(4) Nik1与Cdc28复合物在遗传和生化上相互作用。(5) NIK1可能与CDC6存在遗传相互作用。这些结果表明，Nik1与Cdc28复合物相互作用，不仅在细胞周期的G2/M转变中起作用，而且在细胞周期的S期也起作用。我们分析了nik1 Ej的表型，这些表型被认为是由DNA复制中的一些缺陷引起的，即在s期相关的突变株中，HU敏感性、质粒不稳定性和热敏性增加。这些表型均被swe1 Ej抑制，而Nik1可使swe1磷酸化，因此推测可能是由于S期Cdc28活性降低所致。事实上，对flag标记的clb5依赖性激酶活性的体外分析显示，与clb2依赖性激酶一样，htis的激酶活性受到Swe1的抑制。此外，cdc28Y19E突变体表现出DNA复制的起始和进展延迟。酪氨酸磷酸化的Cdc28出现在S期。这些数据表明，s期CDK复合物，至少包括Clb5-Cdc28复合物，受保守酪氨酸磷酸化的调节。