Summary Broad‐host‐range plasmid RK2 encodes a post‐segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication‐proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase‐ and gyrase‐dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase–DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.

The Escherichia coli protein DnaA and the plasmid RK2-encoded TrfA protein are required for initiation of replication of the broad host range plasmid RK2. The TrfA protein has been shown to bind to five 17-base pair repeat sequences, referred to as iterons, at the minimal replication origin (oriV). Using DNase I footprinting and a gel mobility shift assay, purified DnaA protein was found to bind to four DnaA consensus binding sequences immediately upstream of the five iterons at the RK2 origin of replication. Binding of the TrfA protein to the iterons results in localized strand opening within the A؉T-rich region of the replication origin as determined by reactivity of the top and bottom strands to potassium permanganate (KMnO 4 ). The presence of either the E. coli DnaA or HU protein is required for the TrfA-mediated strand opening. Although the DnaA protein itself did not produce an RK2 open complex, it did enhance and/or stabilize the TrfA-induced strand opening.

The initiation of replication of a plasmid that is able to be maintained in a diverse group of bacteria (broad host range) is considerably less well understood than the initiation of replication of other prokaryotic replicons. It is of particular interest to determine the nature of the interactions between a broad host plasmid origin of replication, the plasmid-encoded replication initiation protein and the host specific replication proteins that are responsible for initiating replication. The broad host range plasmid RK2 requires for its replication in Escherichia coli an origin of replication (oriV) (Fig. 1) and a plasmid encoded initiation protein (TrfA) (1-3) which binds as a monomer to 17-bp 1 direct repeats (iterons) at this origin (4, 5). In addition, RK2 replication in E. coli requires the host specified proteins DnaA, DnaB, DnaC, DNA gyrase, DnaG primase, DNA polymerase III holoenzyme, and SSB (6, 7). It has been shown recently that the E. coli DnaA protein binds to four DnaA consensus sequences that potentially can form a cruciform structure within the RK2 origin (8). In this same study it was found that the TrfA initiation protein in the presence of HU produces an opening of a set of 13-mers located in the A ϩ T-rich region of the RK2 oriV. The DnaA protein enhances and/or stabilizes this open complex formation, but cannot on its own form an open complex.Initiation of replication at the E. coli chromosome origin (oriC) has been studied extensively and the formation of several distinct nucleoprotein complexes has been described (9). The initial step involves binding of the DnaA protein to DnaA boxes localized within oriC (10, 11). This binding results in destabilization of the duplex DNA at the A ϩ T-rich region and open complex formation (12)(13)(14). The DnaB helicase (15) in the form of a DnaB-DnaC complex is specifically loaded at the open region of the origin (16 -18). Cross-linking, enzyme-linked immunosorbent assay, and monoclonal antibody interference studies have shown that a physical interaction between the DnaA protein and the helicase is required for loading (19). An E. coli prepriming complex consisting of oriC DNA and the DnaA, DnaB, DnaC, and HU proteins can be isolated in vitro (19,20). Two stages of prepriming complex formation at oriC have been described (9). During the first stage (prepriming complex I) helicase is loaded but is not active. It has been proposed that the activation of prepriming complex I is the result of the repositioning of the helicase leading to the formation of prepriming complex II. At this stage helicase can unwind template DNA thus allowing the priming reaction to occur.This study utilizes an in vitro replication system reconstituted from purified components to address the mechanism of helicase loading during the initiation of plasmid RK2 replication in E. coli and the role of the DnaA and TrfA proteins in this process. We show that not unlike that observed with oriC (19), a specific interaction between DnaA and DnaB is required for helicase delivery to the in...

Repeated sequences are commonly present in the sites for DNA replication initiation in bacterial, archaeal, and eukaryotic replicons. Those motifs are usually the binding places for replication initiation proteins or replication regulatory factors. In prokaryotic replication origins, the most abundant repeated sequences are DnaA boxes which are the binding sites for chromosomal replication initiation protein DnaA, iterons which bind plasmid or phage DNA replication initiators, defined motifs for site-specific DNA methylation, and 13-nucleotide-long motifs of a not too well-characterized function, which are present within a specific region of replication origin containing higher than average content of adenine and thymine residues. In this review, we specify methods allowing identification of a replication origin, basing on the localization of an AT-rich region and the arrangement of the origin's structural elements. We describe the regularity of the position and structure of the AT-rich regions in bacterial chromosomes and plasmids. The importance of 13-nucleotide-long repeats present at the AT-rich region, as well as other motifs overlapping them, was pointed out to be essential for DNA replication initiation including origin opening, helicase loading and replication complex assembly. We also summarize the role of AT-rich region repeated sequences for DNA replication regulation.

Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.