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 激酶及其激活剂 Dbf4 蛋白最初在芽殖酵母中发现，在真核生物中广泛保守，包括裂殖酵母和人类 Dbf4 相关激活剂（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。）其激酶活性是细胞周期调节的，主要是通过其水平增加的激活亚基的可用性。来自不同真核生物的 Cdc7 调节亚基的氨基酸序列的比较揭示了三小段保守氨基酸序列的存在，即 Dbf4-基序。 N（BRCT 相关）、Dbf4-motif-M 和 Dbf4-motif-C（C2H2 锌指相关）在体外，仅包含 motif-M 的小片段。或基序-C 单独与 Hskl 结合，在体内，仅包含基序-M（113 个氨基酸）和基序-C（61 个氨基酸）的 174 个氨基酸的多肽能够支持 himl null c … More 细胞的有丝分裂生长。以及激酶激活，因此证明 Himl 与 Hskl 的二分结合足以激活激酶及其体内功能，尽管对于有丝分裂功能不是必需的，但可能是相互作用所必需的。 Himl 与染色质。缺乏muCdc7 基因的小鼠在E3.5 和E6.5 之间死亡。条件敲除ES 细胞中muCdc7 功能的失活导致细胞生长快速停滞和DNA 合成停止，随后p53 表达增加和细胞死亡。为了研究小鼠发育中CDK和Cdc7途径之间的相互作用，我们尝试生成muCdc7-/-p27-/-双敲除小鼠。在 E8.5 时检测到存活胚胎，但此后检测不到，表明 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。我们已经表明，Cdks 和 Cdc7 的协同作用对 MCM 的磷酸化可能对于 MCM 的启动很重要。正在对哺乳动物和酵母中含有缺乏潜在磷酸化位点的突变型 MCM2 的复合物进行生化和遗传学表征，以阐明 Cdc7 介导的起源激活的分子基础。