EXPRESSION OF EUKARYOTIC DNA REPLICATION PROTEIN COMPLEXS AND THEIR SIMPLE PURIFICATION METHOD

真核DNA复制蛋白复合物的表达及其简单纯化方法

• 批准号: 07558101
• 负责人: SUGINO Akio
• 金额: $ 13.63万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558101/
• 关键词: DNA Replication protein complex; Expression vector; Baculovirus expression vector; Insect cells; Budding yeast; Cdc7; Dbf4 protein kinase complex; RF-C complex; DNA polymerase complex; YACベクター; Cdc7-Dbf4複合体; RFC複合体

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 … More 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

在过去的一年中，我们已经成功地表达酵母酿酒酵母Cdc 7/Dbf 4蛋白激酶复合物，这是所需的酵母染色体DNA复制的启动使用杆状病毒载体在Sf 9昆虫细胞。本研究以Mcm 2蛋白为激酶底物，尝试建立了一种新的、简便的从含CDC 7和DBF 4基因的重组病毒感染的Sf 9细胞中纯化Cdc 7/Dbf 4蛋白复合物的方法。使用MonoQ、羟基磷灰石和肝素-琼脂糖柱，我们能够纯化蛋白质复合物。使用纯化的Cdc 7/Dbf 4蛋白激酶复合物，我们发现该激酶不仅磷酸化酿酒酵母Mcm 2蛋白，而且磷酸化Mcm 3、Mcm 4、Mcm 6和Dbf 4蛋白。然而，从大肠杆菌中表达和纯化的Mcm蛋白不是该激酶的良好底物，这表明该激酶偏好部分磷酸化的蛋白。 ...更多信息 F-C复合物由5个不同的亚基（Rfc 1 p（Cdc 44 p）、Rfc 2 p、Rfc 3 p、Rfc 4p和Rfc 5 p）组成。在半乳糖的存在下，酵母细胞表达至少50倍以上的RF-C活性比在没有半乳糖。由于PCNA刺激RF-C复合物的ATP酶活性，RF-C复合物与PCNA应紧密结合。使用该柱，我们能够从1 L酵母细胞培养物中获得超过mg的高度纯化的RF-C复合物。这一纯量的RF-C复合物使我们对RF-C复合物、DNA聚合酶和PCNA之间的蛋白质-蛋白质相互作用进行了更详细的研究。最后，我们尝试用酵母表达载体或空泡病毒载体表达酵母DNA聚合酶II和III复合物。然而，到目前为止，我们不能获得任何表达DNA聚合酶复合物的系统。我们不能获得任何表达酵母DNA聚合酶复合物的系统的主要原因是由于大肠杆菌中含有编码DNA聚合酶催化亚基的基因的不稳定的重组载体DNA。少

项目成果

Noskov, V.: "The RFC2 gene encoding the third largest subunit of the RF-C complex is required for chromosomal DNA replication and cell cycle checkpoint in the yeast Saccharomyces cerevisiae." Mol. Cell Biol.17 (in press). (1997)

Noskov, V.：“编码 RF-C 复合体第三大亚基的 RFC2 基因是酿酒酵母中染色体 DNA 复制和细胞周期检查点所必需的。”

Sugimoto, K.: "Rfc5, a small subunit of replicaton factor C complex, couples DNA replication and mitosis in budding yeast." Proc. Natl. Acad. Sci., USA. 93. 7048-7052 (1996)

Sugimoto, K.：“Rfc5 是复制因子 C 复合物的一个小亚基，在芽殖酵母中耦合 DNA 复制和有丝分裂。”

Hashimoto, K.: "The second subunit of DNA polymerse III (δ)is encoded by the HYS2 gene in Saccharomyces cerevisiae." J. Biol. Chem.272 (in press). (1997)

Hashimoto, K.：“DNA 聚合酶 III (δ) 的第二个亚基由酿酒酵母中的 HYS2 基因编码。”J. Biol.272（出版中）。

Hashimoto, K., Ohara, T., Maki, S., and Sugino, A.: "The second subunit of DNA polymerase III (delta) is encoded by the HYS2 gene in Saccharomyces cerevisiae." J.Biol.Chem.(in press). (1997)

Hashimoto, K.、Ohara, T.、Maki, S. 和 Sugino, A.：“DNA 聚合酶 III (δ) 的第二个亚基由酿酒酵母中的 HYS2 基因编码。”

Sugimoto,K.: "Rfc5,a small subunit of replication factor C complex,couples DNA replication and mitosis in budding yeast" Proc.Natl.Acad.Sci.,USA. 93. 7048-7052 (1996)

Sugimoto,K.：“Rfc5，复制因子 C 复合体的一个小亚基，在芽殖酵母中耦合 DNA 复制和有丝分裂”Proc.Natl.Acad.Sci.，美国。