| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
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.
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