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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

我们一直在广泛研究G1/S转换的分子机制及其调控。1）Cdc 7激酶及其激活因子Dbf 4蛋白最初在芽殖酵母中发现，在真核生物（包括裂殖酵母和人类）中具有广泛的保守性。通过构建条件性敲除ES细胞，我们证明Cdc 7的功能对哺乳动物细胞的DNA复制和增殖活动是必需的。2）比较不同真核生物Cdc 7调控亚基的氨基酸序列，发现存在三个小的保守氨基酸序列，即Dbf 4-motif-N（BRCT相关）、Dbf 4-基序-M和Dbf 4-基序-C（C2 H2锌指相关）。在体外，含有基序-M单独或基序-C单独的小片段结合Hsk 1。在体内，一个174个氨基酸的多肽，只含有基序-M（113个氨基酸）和基序-C（61个氨基酸），能够支持有丝分裂生长的h ...更多信息 im 1无效细胞以及激酶活化，从而证明Him 1与Hsk 1的二分结合足以用于激酶活化及其在体内的功能。基序-N，虽然不是有丝分裂功能所必需的，但可能是Him 1与染色质相互作用所必需的。3）缺乏muCdc 7基因的小鼠在E3.5和E6.5之间死亡。为了研究小鼠发育中CDK和Cdc 7通路之间的相互作用，我们尝试产生muCdc 7-/- p27-/-双敲除小鼠。在E8.5检测到有活力的胚胎，但此后没有，表明CDK活性的增加可以部分挽救muCdc 7-/-胚胎的早期胚胎生长。4）我们已经在人染色体5 q上的IL-3/GM-CSF细胞因子簇区域中定位了复制起点，该区域紧邻GM-CSF基因的下游。此外，我们发现，ORC，Cdc 6和MCM蛋白特异性结合到这个区域。通过与已知的后生动物复制起点序列的比较，我们确定了一个可能的共有序列。少