DnaA box sequences as the site for helicase delivery during plasmid RK2 replication initiation in Escherichia coli

DnaA box sequences are a common motif present within the replication origin region of a diverse group of bacteria and prokaryotic extrachromosomal gen etic elements. Although the origin opening caused by binding of the host Dn aA protein has been shown to be critical for the loading of the DnaB helica se, to date there has been no direct evidence presented for the formation o f the DnaB complex at the DnacA box site. For these studies, we used the re plication origin of plasmid RK2 (oriV), containing a cluster of four DnaA b oxes that bind DnaA proteins isolated from different bacterial species (Cas pi, R., Helinski, D. R., Pacek, M,, and Konieczny, I. (2000) J. Biol. Chem. 275, 18454-18461). Size exclusion chromatography, surface plasmon resonanc e, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized similar to 200 base pairs upstream hom the region of Origin opening and a potential site for h elicase entry. The DnaABC complex was formed on both double-stranded superh elical and linear RK2 templates. A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed. In additio n, our experiments provide evidence for interaction between the plasmid ini tiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region. This interaction is facilitated via direct contact between TrfA and DnaB proteins.