Abstract
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.
Highlights
The initiation of DNA replication of prokaryotic and eukaryotic replicons requires delivering and loading of a helicase at the replication origin [2, 3]
Our results demonstrate for the first time the primary formation of an E. coli DnaB helicase complex at the DnaA box region of a plasmid replication origin
These results are consistent with the original studies on DNA replication initiation at the E. coli replication origin oriC [2, 48]
Summary
The initiation of DNA replication of prokaryotic and eukaryotic replicons requires delivering and loading of a helicase at the replication origin [2, 3]. The binding of DnaA to DnaA boxes at the E. coli chromosomal replication origin results in destabilization of duplex DNA at the AϩT-rich region and an open complex formation [4, 23,24,25,26]. The 393-base pair (bp) minimal origin, oriV, from the plasmid RK2, contains five 17-bp direct repeats [42], four DnaA boxes, and AϩT-rich repeat sequences (13-mers), which are the initial sites of helix destabilization (13 )(Fig. 1). It has been demonstrated that the plasmid RK2-specific replication initiation protein, TrfA, binds to the iteron sequences and, in the presence of ATP and HU and/or DnaA, produces an opening of a set of 13-mers within the AϩT-rich region [13]. As for oriC [31], a specific interaction between the DnaA and DnaB proteins has been shown; both DnaA and TrfA proteins are required for helicase activity at RK2 DNA [43]
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