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

DNA复制是由一个叫做复制体的大分子复合体完成的，复制体是由叉状结构的DNA和许多蛋白质因子组成的。本研究的目的是了解复制体复合体的组装和重组的分子机制，为精确的DNA复制过程奠定基础。我们的研究的创新之处在于我们使用了高温古生菌，它提供了非常有用的实验材料，特别适合于络合物的结构分析。我们鉴定了与染色体DNA的复制起点(ORIC)特异结合的CDC6/ORC1蛋白。利用电子显微镜分析，我们观察了该蛋白的结构性质，发现该蛋白的结构转换依赖于核苷酸辅助因子。此外，我们还对我们自己从古细菌细胞中发现的Hef和HJm蛋白进行了鉴定。这些蛋白质通过将复制的DNA的叉状结构转换成适合于随后的重组修复过程的形式，来工作于与DNA复制相结合的修复系统。Kohda博士是这项研究的合作者之一，他已经阐明了PriA可能是大肠杆菌中HeF的功能同源物，具有识别复制叉中新生DNA末端的能力，并为这种识别机制提供了结构基础。

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.