Helicase Delivery and Activation by DnaA and TrfA Proteins during the Initiation of Replication of the Broad Host Range Plasmid RK2

Abstract

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...

Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly

“…The Pol III core, however, was not required, and the absence of gyrase reduced β-clamp complex formation only slightly. The absence of DnaA in this complex results in an inefficient origin opening by TrfA (26); disturbances in helicase recruitment (28); and, as we demonstrate in this work, defects in β-clamp delivery on plasmid DNA. These observations support an indirect involvement of DnaA in replisome assembly.…”

“…2A). In addition to supercoiled DNA, TrfA variants, and DnaB, the reaction mixture contained DnaA and DnaC proteins, which are required for helicase complex formation at oriV (28). Under these experimental conditions, plasmid DNA was found in the column's void volume fractions ( Fig.…”

“…We therefore tested the TrfA variants as helicase-activating factors. E. coli DnaB helicase activity on supercoiled RK2 plasmid DNA template was identified electrophoretically by the formation of an extensively unwound form of the supercoiled plasmid DNA, designated FI* (28). The formation of the FI* form is a result of the combined activities of plasmid and bacterial Reps, with the presence of an active form of TrfA being essential.…”

“…S3D). Because TrfA is involved in DnaB helicase complex formation at oriV (27,28), we decided to test if the TrfA ΔLF and TrfA F138A, which were defective in replication, were able to recruit the helicase. Using gel filtration on a Sepharose CL-4B column (GE Healthcare), we tested helicase complex formation on a supercoiled DNA template containing oriV (pKD19L1) in the presence of ClpX-activated TrfA variants ( Fig.…”

“…TrfA interaction with the iterons leads to origin opening assisted by host HU and DnaA proteins (26). TrfA plays a crucial role in DnaB helicase recruitment and positioning at the AT-rich region of the oriV (27,28). In contrast to DnaA protein, no data for plasmid Rep filament formation on ssDNA have been provided to date.…”