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 genetic elements. Although the origin opening caused by binding of the host DnaA protein has been shown to be critical for the loading of the DnaB helicase, to date there has been no direct evidence presented for the formation of the DnaB complex at the DnaA box site. For these studies, we used the replication origin of plasmid RK2 (oriV), containing a cluster of four DnaA boxes that bind DnaA proteins isolated from different bacterial species (Caspi, R., Helinski, D. R., Pacek, M., and Konieczny, I. (2000) J. Biol. Chem. 275, 18454-18461). Size exclusion chromatography, surface plasmon resonance, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized approximately 200 base pairs upstream from the region of origin opening and a potential site for helicase entry. The DnaABC complex was formed on both double-stranded superhelical and linear RK2 templates. A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed. In addition, our experiments provide evidence for interaction between the plasmid initiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region. This interaction is facilitated via direct contact between TrfA and DnaB proteins.
Pacek
M
M
Faculty of Biotechnology, Department of Molecular and Cellular Biology and the Faculty of Biology, Department of Molecular Biology, University of Gdansk, 24 Kladki, PL-80822 Gdansk, Poland.
Konopa
G
G
Konieczny
I
I
D001426:Bacterial Proteins / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism* / Q000648:ultrastructure; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000648:ultrastructure; D053488:DnaB Helicases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C067405:TrfA protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases