MASAI Hisao UNIV.TOKYO,INSTIT.MED.SCI., ASSOCIATE PROFESSOR, 医科学研究所, 助教授 (40229349)
TSURIMOTO Toshiki NARA INSTIT.SCI.TECH., ASSOCIATE PROFESSOR, バイオサイエンス科, 助教授 (30163885)
ITOH Tateo SHINSHU UNIV., FACULTY OF SCIENCE,PROFESSOR, 理学部, 教授 (40051817)
MAKI Hisaji NARA INSTIT.SCI.TECH,PROFESSOR, バイオサイエンス科, 教授 (20199649)
MAKI Satoko OSAKA UNIV.RES.INSTIT.FOR MICROBIAL DISEASES,RESEARCH ASSOCIATE, 微生物病研究所, 助手 (60212205)
|Budget Amount *help
¥21,300,000 (Direct Cost: ¥21,300,000)
Fiscal Year 1996: ¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1995: ¥14,300,000 (Direct Cost: ¥14,300,000)
In last year, we have succeeded in expressing yeast Saccharomyces cerevisae Cdc7/Dbf4 protein kinase complex which is required for the initiation of yeast chromosomal DNA replication using Vaculovirus vector in Sf9 insect cells. In this year, we tried to develop a new and simple purification method of Cdc7/Dbf4 protein complex from Sf9 cells infected with the recombinant virus containing CDC7 and DBF4 genes using Mcm2 protein as a kinase substrate. Using MonoQ,hydroxylapatite, and Heparin-Sepharose columns, we were able to purify the protein complex. Using the purified Cdc7/Dbf4 protein kinase complex, we found that the kinase phosphorylates not only S.cerevisiae Mcm2 protein, but also Mcm3, Mcm4, Mcm6, and Dbf4 protein. However, Mcm proteins expressed in and purified from E.coli were not good substrates for the kinase, suggesting that the kinase prefers partially phosphorylated proteins.
Using yeast expression vector containing Gal 1 and Gal10 promotors, we were able to express yeast R
F-C complex consisting of five different subunits (Rfc1p (Cdc44p), Rfc2p, Rfc3p, Rfc4p and Rfc5p) in yeast. In the presence of galactose, yeast cells expressed at least 50 time more RF-C activity than in the absence of galactose. Since PCNA stimulates the ATPase activity of RF-C complex, RF-C complex should tightly interact with PCNA.Based un this, we made PCNA-conjugated Sepharose column. Using this column, we were able to obtain more than mg of highly purified RF-C complex from 1 L culture of yeast cells. This amount of pure RF-C complex makes more detailed study on protein-protein interaction between RF-C complex, DNA polymerases and PCNA.
Finally, we tried to express yeast DNA polymerases II and III complex using either yeast expression vectors or vaculovirus vector. However, so far we were not able to obtain any system expressing DNA polymerase complex. The main reason why we could not obtain any system that expresses yeast DNA polymerase complex was due to unstable recombinant vector DNA containing the gene encoding the DNA polymerase catalytic subunit in E.coli. Less