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

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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.