Protein Dynamics in Complex Apparatus for the Initiation and Regulation of the Chromosomal Replication

染色体复制启动和调节的复杂装置中的蛋白质动力学

基本信息

批准号：
16370081
负责人：
KATAYAMA Tsutomu
金额：
$ 8.64万
依托单位：
KYUSHU UNIVERCITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

DnaA protein promotes the initiation reaction in the complex with the replication origin. In this process, the duplex DNA within the origin is unwound. Also, DnaA is a crucial target of systems to regulate replicational initiation during the cell cycle. In this research project, we had a specific aim to analyze the mechanisms in the replicational initiation and its regulatory systems from a view of dynamic and comprehensive interprotein interactions.1 Structure analysis of DnaA and the initiation complexWe analyzed a hyperthermophilic eubacterium DnaA homolog and succeeded in construction of in vitro reconstituted system for the replication initiation. We promoted production of crystals of the initiation complex using this DnaA homolog. Also, we analyzed the function-structure relationship in E. coli DnaA N-terminal domains and revealed the mechanisms in inter-DnaA interaction and DnaA-DnaB helicase interaction.2 Interaction of DnaA, the replicative clamp, and HdaWe found the function-structure relationship of Hda in homooligomer formation. Also, we analyzed the function-structure relationship of the clamp in interaction with Hda. Moreover, we used an advanced proteomix method to search for novel factors that interact with the clamp, resulting in identification of many candidates. We further analyzed these factors.3 Analysis of a novel DnaA-associating factorWe isolated many mutant forms of DiaA, a novel DnaA-associating protein and analyzed the function-structure relationship. Also, we revealed the crystal structure of DiaA. Moreover, we for the first time revealed that DiaA enhances the process of the initiation complex formation.4 Design and chemical synthesis of specific inhibitors for DnaA DNA-binding domainWe designed and synthesized oligonucleotides that were chemically modified to covalently bind to DnaA DNA-binding domain. Binding kinetics of these chemicals was analyzed.

DNAA蛋白促进复合物与复制起源的起始反应。在此过程中，原点内的双链DNA已解开。同样，DNAA是调节细胞周期复制起始的系统的关键目标。在该研究项目中，我们有一个具体的目的是从动态和全面的诠释蛋白相互作用的角度分析复制启动及其调节系统的机制。1DNAA的结构分析和Initiation Complextialwe分析了高疗菌Eubacterium DNAA同源性的高疗菌Eubacterium dnaa同源性，并成功地构建了重新定位的复制系统。我们使用此DNAA同源物促进了起始复合物的晶体。此外，我们分析了大肠杆菌DNAA N末端结构域中的功能结构关系，并揭示了DNAA相互作用和DNAA-DNAB解旋酶相互作用的机制。2DNAA的相互作用，复制性夹具和HDAWE的相互作用发现了HOMILIMIGIGOMER形成中HDA的功能结构关系。另外，我们分析了夹具与HDA相互作用的功能结构关系。此外，我们使用了先进的蛋白质组分方法来搜索与夹具相互作用的新因素，从而识别了许多候选者。 3分析新型DNAA缔解因子We分离了多种突变形式的Diaa，这是一种新型的DNAA缔解蛋白，并分析了功能结构关系。另外，我们揭示了Diaa的晶体结构。此外，我们首次揭示了DIAA增强了起始复合物的过程。4设计和化学合成DNAA DNAA DNA结合域的特定抑制剂设计和合成化学修饰以共价结合DNAA DNA DNA结合结构域。分析了这些化学物质的结合动力学。