Joint study on DNA replication and checkpoint control

DNA复制和检查点控制的联合研究

基本信息

批准号：
11694247
负责人：
ARAI Ken-ichi
金额：
$ 10.75万
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 (A)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

We have been extensively studying the molecular mechanisms of G1/S transition and its regulation. The results obtained from these studies can be summarized as follows1) Cdc7 kinase and its activator Dbf4 protein, originally identified in budding yeast are widely conserved in eukaryotes including fission yeast and human. We have demonstrated that Cdc7 functions are essential for DNA replication and proliferation activities of mammalian cells by generating conditional knockout ES cells.2) 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 Hsk1. 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 h … More im1 null cells as well as kinase activation, thus demonstrating that bipartite binding of Him1 to Hsk1 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 Him1 with chromatin.3) Mice lacking muCdc7 genes die between E3.5 and E6.5. 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.4) We have located a replication origin in the IL-3/GM-CSF cytokine cluster region on the human chromosome 5q at the region immediately downstream of GM-CSF gene. Furthermore, we showed that ORC, Cdc6 and MCM proteins are specifically bound to this region. Through comparison with other known metazoan replication origin sequences, we have identified a possible consensus sequence. Less

我们已经研究了G1/Sumar的分子机制，如下1）CDC7激酶及其激活剂DBF4蛋白，最初在芽中鉴定出在真核生物Incllion AST和人类中广泛保守的cdc7功能，我们证明了CDC7功能对于DNA复制和成果的必不可少。哺乳动物细胞通过Genditi Onal敲除ES细胞。2）三个小的保守氨基酸序列，即DBF4-MOTIF-N（与BRCT相关），DBF4-MOTIF-M和DBF4-MOTIF-C（C2H2 ZINC相关的DBF4-MOTIF-M））TRO，一个单独包含基序m或单独的基序C的小节与HSK1结合，A174氨基酸多肽（113个氨基酸）和基序C（61氨基酸）能够移植H …更多的IM1无效细胞和激酶激活，因此，HIM1与HSK1的结合对于激酶ATS在体内的功能是令人吃惊的。缺乏MUCDC7基因在E3.5之间死亡。 CDK活性可以部分挽救MUCDC7 - / - 胚胎的早期Emryon，4）在GM-CSF基因下游的人类染色体5Q上的IL-3/GM-CSF细胞因子簇区域中的eplication起源表明兽人，cdc6和mcm蛋白与该区域的粗糙比较特别结合