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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.

Dna A蛋白促进具有复制起点的复合体中的起始反应。在这个过程中，原点内的双链DNA被解开。此外，DNAA也是系统在细胞周期中调节复制启动的关键靶点。在本研究项目中，我们有针对性地从动态和全面的蛋白质相互作用的角度来分析复制起始及其调控系统的机制。1.Dna A和起始复合体的结构分析我们分析了一个嗜热的真细菌Dna A同源物，并成功地构建了复制起始的体外重组系统。我们利用这个DNAA同系物促进了引发复合体晶体的生产。此外，我们还分析了大肠杆菌Dna A N端区的功能结构关系，揭示了Dna A间相互作用和Dna A-Dna B解旋酶相互作用的机制。2.Dna A、复制夹子和Hda的相互作用。此外，我们还分析了夹子与HDA相互作用时的功能-结构关系。此外，我们使用一种先进的蛋白质组学方法来寻找与钳子相互作用的新因子，从而鉴定出许多候选基因。我们分离了一种新的DNAA结合蛋白DiaA的多种突变形式，并对其功能结构关系进行了分析。此外，我们还揭示了DiaA的晶体结构。4 DNAA DNA结合域特异性抑制剂的设计与化学合成我们设计并合成了经化学修饰后与DNAA DNA结合域共价结合的寡核苷酸。对这些化合物的结合动力学进行了分析。