Replication initiation at a distance: determination of the cis- and trans-acting elements of replication origin alpha of plasmid R6K

Abstract

Plasmid R6K, which contains 3 replication origins called alpha, gamma, and beta, is a favorable system to investigate the molecular mechanism(s) of action at a distance, i.e. replication initiation at a considerable distance from the primary initiator protein binding sites (iterons). The centrally located gamma origin contains 7 iterons that bind to the plasmid-encoded initiator protein, pi. Ori alpha, located at a distance of approximately 4 kb from gamma, contains a single iteron that does not directly bind to pi but is believed to access the protein by pi-mediated alpha-gamma iteron-iteron interaction that loops out the intervening approximately 3.7 kb of DNA. Although the cis-acting components and the trans-acting proteins required for ori gamma function have been analyzed in detail, such information was lacking for ori alpha. Here, we have identified both the sequence elements located at alpha and those at gamma, that together promoted alpha activity. The data support the conclusion that besides the single iteron, a neighboring DNA primase recognition element called G site is essential for alpha-directed plasmid maintenance. Sequences preceding the iteron and immediately following the G site, although not absolutely necessary, appear to play a role in efficient plasmid maintenance. In addition, while both dnaA1 and dnaA2 boxes that bind to DnaA protein and are located at gamma were essential for alpha activity, only dnaA2 was required for initiation at gamma. Mutations in the AT-rich region of gamma also abolished alpha function. These results are consistent with the interpretation that a protein-DNA complex consisting of pi and DnaA forms at gamma and activates alpha at a distance by DNA looping.

FIGURE 1.

Schematic depiction of α-γ and mini α-γ replicons and a model of a preinitiation complex at α. A, schematic diagram showing the full α-γ sequence, the locations of the various iterons and half-iterons are shown. B, mini α-γ replicon formed by retaining 660 bp from the left end and deleting the remainder of the sequence all the way to, but not including the dnaA1 box of γ. C, model of a preinitiation complex formed at ori α, showing the 6 iterons of γ postulated to be wrapped around the monomeric π protein molecules, the single α iteron bound to a monomeric π and paired with the γ iteron (also bound to a monomeric π with the iteron pair stabilized by a dimeric π), and possibly a pair of DnaA proteins bound to the dnaA1 and dnaA2 boxes and containing a melted region.

FIGURE 2.

Diagrams showing the locations of various deletions of the α sequence of a mini α-γ replicon and their relative efficiencies with regard to plasmid maintenance in vivo in IHF⁺ and ihfΔ hosts. A, nucleotide sequence of the mini α region, showing the βHR, iteron, and G site that are boxed, the presumptive start point of DnaG-catalyzed primer RNA, and the nick site belonging to the closely located oriT. B, schematic representations of the various deletions and their effect on α-γ plasmid maintenance. C, quantitative analysis of plasmid maintenance in replicons deleted for various sequences about ori α in both IHF⁺ and the ihfΔ hosts on 50 and 100 μg/ml ampicillin plates. The asterisks indicate that no colonies were formed by host cells containing the particular plasmid derivatives.

FIGURE 3.

SS to DS replication in vitro of DNA templates carrying the wt R6K G site, the origin of G4 (M13 G ori1), and the two mutant forms of the plasmid G site. A, sequences showing the wt and the Not1 and the CAG mutant forms; the bottom line shows the start point of the presumptive primer RNA. B, diagram showing the purified proteins used and their action in replicating SS to DS DNA. C, quantification of the reaction product from each template from three separate experiments, presented with S.E. bars. D, in vivo measurements of maintenance of amp-resistant IHF⁺ and ihfΔ hosts carrying the wt and the various G site mutants along with a pUC19 control plated on LB media containing 40 μg/ml ampicillin.

FIGURE 4.

Replication of α-γ and mini α-γ plasmids analyzed by Brewer-Fangman two-dimensional gel electrophoresis. A, schematic diagrams of the α-γ and the mini α-γ templates plasmids used. B, schematic representation of the expected two-dimensional gel pattern after probing with an α-specific labeled DNA probe of gels blotted onto nitrocellulose membranes showing the bubble, the Y, and the double Y arcs. C, replication intermediates of full-length α-γ plasmid showing a fainter bubble arc and strong Y and double Y arcs. D, replication intermediates of the mini α-γ plasmid showing a pattern very similar to that in C.

FIGURE 5.

Mutants of ori γ sequences that impact ori α-mediated plasmid maintenance. A, diagram showing the normal and the mutated γ sequences. (+) means at least 500 colonies of IHF⁺ and/or ihfΔ cells carrying the plasmid derivatives plated on 40 μg/ml amp plates were recorded. (−) means that the ihfΔ host carrying the various plasmid derivatives formed less than 1% of the colonies formed by the IHF⁺ cells carrying the same plasmid derivative under identical conditions. Data are averages of three independent sets of experiments. The wt and the mutant form of the AT-rich region with the embedded ihf site are shown; the Xho1 insertion precisely separated the adjacent 7th iteron and the DnaA2 box. B, gel mobility shift experiments performed with 20 fmol of labeled ori γ probe/reaction and containing the wt or the mutated ihf site and incubated with the indicated amounts (fmol) of purified IHF. The mutant form of the ihf site failed to bind to the protein as contrasted with the normal ihf site.