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 蛋白促进与复制起点复合物中的起始反应。在此过程中，起点内的双链 DNA 被解开。此外，DnaA 是细胞周期中调节复制起始的系统的重要靶标。在本研究项目中，我们的具体目标是从动态和全面的蛋白质间相互作用的角度分析复制起始及其调节系统的机制。 1 DnaA 和起始复合物的结构分析我们分析了超嗜热真细菌 DnaA 同源物，并成功地构建用于复制起始的体外重构系统。我们利用这种 DnaA 同系物促进了起始复合物晶体的生产。此外，我们还分析了大肠杆菌DnaA N端结构域的功能-结构关系，揭示了DnaA间相互作用和DnaA-DnaB解旋酶相互作用的机制。2 DnaA、复制钳和Hda的相互作用我们发现了功能-结构Hda 在同源低聚物形成中的关系。此外，我们还分析了夹子与 Hda 相互作用的功能结构关系。此外，我们使用先进的蛋白质组合方法来寻找与钳相互作用的新因子，从而鉴定出许多候选因子。我们对这些因素进行了进一步的分析。3新型DnaA相关因子的分析我们分离了新型DnaA相关蛋白DiaA的多种突变形式，并分析了其功能与结构的关系。此外，我们还揭示了 DiaA 的晶体结构。此外，我们还首次揭示了DiaA增强了起始复合物形成的过程。4 DnaA DNA结合域特异性抑制剂的设计和化学合成我们设计并合成了经过化学修饰的寡核苷酸，使其与DnaA DNA结合域共价结合。。分析了这些化学物质的结合动力学。