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Structural and functional analyses of the DNA replication machinery called replisome complex

DNA 复制机制（称为复制体复合体）的结构和功能分析

基本信息

批准号：
15310151
负责人：
ISHINO Yoshizumi
金额：
$ 9.86万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

DNA replication processes by a macromolecular complex called replisome, which was made of fork-structured DNA and many protein factors. The aim of this research is to understand the molecular mechanism of assembling and reorganizing of the replisome complex for the precise progress of DNA replication. The originarity of our esearch is that we use hyperthermophilic archaea, which provide very useful experimental materials especially appropriate for the structural analyses of the complexes. We characterized the Cdc6/Orc1 protein that specifically binds to the replication origin (oriC) of the chromosome DNA. Using electron microscopic analyses, we observed the structural properties of this proein, and found that this protein converts its structure depending on the nucleotide cofactors. In addition, we characterized Hef, and Hjm proteins, which were discovered from archaeal cells (Pyrococcus furiosus) by ourselves. These proteins works for the repair systems coupled with DNA replication by converting the fork-structure of replicating DNA to the appropriate form for the following recombinational repair process. Dr.Kohda, a collaborator of this research, has been elucidated that the PriA, which may be the functional homolog of Hef in Esherichia coli, has a recognizing ability of the terminus of the nascent DNA in the replication fork, and provided a structural basis of this recognition mechanism.

DNA复制是通过复制体这种由叉状结构的DNA和多种蛋白质因子组成的大分子复合体进行的。本研究的目的是了解复制体复合物组装和重组的分子机制，以精确地进行DNA复制。本实验的独创性在于我们使用了超嗜热古菌，这为配合物的结构分析提供了非常有用的实验材料。我们的特点是Cdc 6/Orc 1蛋白特异性结合到染色体DNA的复制起点（oriC）。利用电子显微镜分析，我们观察了这种蛋白质的结构特性，并发现这种蛋白质依赖于核苷酸辅因子转换其结构。此外，我们的特点是Hef，和Hjm蛋白，这是我们自己发现的古细菌细胞（Pyrococcus furiosus）。这些蛋白质通过将复制DNA的叉状结构转化为合适的形式用于随后的重组修复过程来为与DNA复制偶联的修复系统工作。本研究的合作者Kohda博士阐明了可能是大肠杆菌Hef功能同系物的PriA对复制叉中新生DNA的末端具有识别能力，并为这种识别机制提供了结构基础。