Functional analysis based on the structure of DNA replication proteins in Escherichia coli

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Based on the structure, we examined the functions of proteins which were associated with DNA replication initiation. Firstly, we examined the N-terminal domain of DnaA. DnaA protein is a key protein in the initiation of chromosomal replication in Escherichia coli. The structure of E. coli DnaA is subdivided into four functional domains. Although the structure of domain III and IV were already determined, the structure of N-terminal domain, which contains DnaA oligomerization activity and DnaB helicase binding sites, was not understood. To determined the structure of N-terminal domain, we assigned the ^1H,^<13>C and ^<15>N backbone resonances of N-terminal domain of Dna A (Bio NMR assignment 2007). Furthermore, we determined the N-terminal domains (1-108) structure using NMR spectroscopic method. Domain I has an α-α-β-β-α-β motif, similar to that of the K homology (KH) domain and has weak affinity for oriC single-stranded DNA, consistent with KH domain function. A hydrophobic surface carrying Trp-6 most likely forms the interface for domain I dimerization. Glu-21 is located on the opposite surface of domain I from the Trp-6 site and is crucial for DnaB helicase loading. These findings suggested a model for DnaA homomultimer formation and DnaB helicase loading on oriC (J. Biol. Chem. 2007) . This paper was selected as Papers of the Week in J. Biol. Chem. 2007, June 15. And then, we examined the protein functional and structural analyses of heat shock protein HSPQ associated with DnaA degradation, cell division re-activator CedA, and the primosome component PriB. Some part of these results was announced at the several academic conferences.