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Studies on regulation of mitotic DNA replication and meiosis by novel Cdc7-related kinase complexes

新型Cdc7相关激酶复合物调控有丝分裂DNA复制和减数分裂的研究

基本信息

批准号：
10480164
负责人：
ARAI Ken-ichi
金额：
$ 9.15万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

Cdc7 kinase and its activator Dbf4 protein, originally identified in budding yeast are widely conserved in eukaryotes including fission yeast and human. Dbf4-related activators (Dfp1/Him1 and ASK) bind and stimulate kinase activity of Cdc7-encoded catalytic subunits (Hsk1 and huCdc7). Its kinase activity is cell cycle-regulated, mainly through availability of the activation subunit whose level increases at the Gl/S boundary and is maintained at a high level throughout S phase. Comparison of the amino acid sequences of the Cdc7-regulatory subunits from various eukaryotes revealed the presence of three small stretches of conserved amino acid sequences, namely Dbf4-motif-N (BRCT-related), Dbf4-motif-M, and Dbf4-motif-C (C2H2 zinc finger-related). In vitro, a small segment containing motif-M alone or motif-C alone binds to Hskl. In vivo, a 174 amino acid polypeptide containing only motif-M (113 amino acids) and motif-C (61 amino acids) is capable of supporting mitotic growth of himl null c … More ells as well as kinase activation, thus demonstrating that bipartite binding of Himl to Hskl is sufficient for kinase activation and for its functions in vivo. Motif-N, although not essential for mitotic functions, may be required for interaction of Himl with chromatin.Mice lacking muCdc7 genes die between E3.5 and E6.5. Inactivation of muCdc7 functions in conditional knockout ES cells resulted in rapid arrest of cell growth and cessation of DNA synthesis, followed by increase of p53 expression and cell death. In order to examine interactions between CDK and Cdc7 pathways in mouse development, we tried to generate muCdc7-/-p27-/-double knockout mice. Viable embryos were detected at E8.5, but not thereafter, indicating that increase of CDK activity can partially rescue the early embryonic growth of muCdc7-/-embryos. MCM2 protein is among physiologically important substrates of Cdc7 kinase. Multiple residues on MCM2 are phosphorylated by Cdc7 in vivo and in vitro. We have shown that phosphorylation of MCM by concerted actions of Cdks and Cdc7 may be important for initiation. MCM complexes containing mutant MCM2 lacking potential phosphorylation sites are being biochemically and genetically characterized in mammals and yeast in order to clarify molecular basis of Cdc7-mediated origin activation. Less

Cdc7激酶及其激活因子Dbf4蛋白最初在出芽酵母中发现，在真核生物包括裂变酵母和人类中广泛保守。dbf4相关激活因子（Dfp1/Him1和ASK）结合并刺激cdc7编码的催化亚基（Hsk1和huCdc7）的激酶活性。其激酶活性受细胞周期调节，主要通过激活亚基的可用性，其水平在Gl/S边界处增加，并在整个S期保持在高水平。比较不同真核生物cdc7调控亚基的氨基酸序列，发现存在3个小片段的保守氨基酸序列，即Dbf4-motif-N （brct相关）、Dbf4-motif-M和Dbf4-motif-C （C2H2锌指相关）。在体外，仅含有基序- m或基序- c的小片段与Hskl结合。在体内，一个174个氨基酸的多肽只含有基元- m（113个氨基酸）和基元- c（61个氨基酸），能够支持himl的有丝分裂生长和激酶激活，从而证明himl与Hskl的两部分结合足以激活激酶及其在体内的功能。基序n虽然不是有丝分裂功能所必需的，但可能是Himl与染色质相互作用所必需的。缺乏muCdc7基因的小鼠在E3.5和E6.5之间死亡。在条件敲除的ES细胞中，muCdc7功能失活导致细胞生长迅速停止，DNA合成停止，随后p53表达增加和细胞死亡。为了研究CDK和Cdc7通路在小鼠发育过程中的相互作用，我们试图产生muCdc7-/-p27-/-双敲除小鼠。在E8.5时可检测到活胚，之后则无法检测到活胚，说明CDK活性的增加可以部分挽救muCdc7-/-胚胎的早期胚胎生长。MCM2蛋白是Cdc7激酶的重要生理底物之一。MCM2上的多个残基在体内和体外被Cdc7磷酸化。我们已经证明Cdks和Cdc7协同作用下MCM的磷酸化可能对起始很重要。为了阐明cdc7介导的起源激活的分子基础，在哺乳动物和酵母中对含有缺乏潜在磷酸化位点的突变体MCM2的MCM复合物进行了生化和遗传学表征。少