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

Abstract

Although the molecular basis for replisome activity has been extensively investigated, it is not clear what the exact mechanism for de novo assembly of the replication complex at the replication origin is, or how the directionality of replication is determined. Here, using the plasmid RK2 replicon, we analyze the protein interactions required for Escherichia coli polymerase III (Pol III) holoenzyme association at the replication origin. Our investigations revealed that in E. coli, replisome formation at the plasmid origin involves interactions of the RK2 plasmid replication initiation protein (TrfA) with both the polymerase β-and α-subunits. In the presence of other replication proteins, including DnaA, helicase, primase and the clamp loader, TrfA interaction with the β-clamp contributes to the formation of the β-clamp nucleoprotein complex on origin DNA. By reconstituting in vitro the replication reaction on ssDNA templates, we demonstrate that TrfA interaction with the β-clamp and sequence-specific TrfA interaction with one strand of the plasmid origin DNA unwinding element (DUE) contribute to strand-specific replisome assembly. Wild-type TrfA, but not the TrfA QLSLF mutant (which does not interact with the β-clamp), in the presence of primase, helicase, Pol III core, clamp loader, and β-clamp initiates DNA synthesis on ssDNA template containing 13-mers of the bottom strand, but not the top strand, of DUE. Results presented in this work uncovered requirements for anchoring polymerase at the plasmid replication origin and bring insights of how the directionality of DNA replication is determined.DNA replication initiation | polymerase III | β-clamp | Rep | plasmid RK2

Replisome Assembly at Bacterial Chromosomes and Iteron Plasmids

“…Then, the δ subunit of clamp loader opens the β-clamp and clamp loader positions it onto primer-template junction, as it is thought to occur during replisome assembly at E. coli oriC . This model is consistent with the results of the in vitro DNA replication experiments performed with the use of ssDNA, containing sequence of RK2 plasmid oriV (Wawrzycka et al, 2015 ). It was demonstrated that TrfA interacts with specific strand of ssDNA of DUE, i.e., the bottom strand, which serves as the site for replisome assembly (Wegrzyn et al, 2014 ; Wawrzycka et al, 2015 ).…”

“…This model is consistent with the results of the in vitro DNA replication experiments performed with the use of ssDNA, containing sequence of RK2 plasmid oriV (Wawrzycka et al, 2015 ). It was demonstrated that TrfA interacts with specific strand of ssDNA of DUE, i.e., the bottom strand, which serves as the site for replisome assembly (Wegrzyn et al, 2014 ; Wawrzycka et al, 2015 ). It can be further speculated that TrfA may assist the clamp loader in recognition of the 3′ end of primer-template junction within the oriV .…”

“…The TrfA ΔLF mutant facilitated the determination of biological relevance of this interaction. The complex of TrfA and β-clamp was found to be the key feature for replisome assembly and thereby for oriV- dependent DNA replication of both supercoiled dsDNA plasmid and ssDNA plasmid in vitro , albeit the clamp loader complex is still crucial (Wawrzycka et al, 2015 ). Hence, the question arises—how do the Rep and clamp loader cooperate to load the β-clamp at plasmid origin ?…”

Defining the crucial domain and amino acid residues in bacterial Lon protease for DNA binding and processing of DNA-interacting substrates

“…Binding of Lon WT, LonK371E/K376E/R379E, and LonR306E/K308E/K310E/K311E to TrfA was analyzed by ELISA as described previously (66) with the following modifications. 200 nM TrfA and 1% (w/v) BSA solution (negative control) were immobilized on the 96-well ELISA plate (Costar) for 90 min.…”

Evolved plasmid‐host interactions reduce plasmid interference cost

“…During initiation of replication, the initiator proteins of iteron‐containing plasmids like those of the IncP‐1 group need to interact with host proteins such as DnaA, DnaB, sliding clamp and protein chaperones (DnaKJ/GrpE, ClpB, ClpX, ClpA) (Konieczny et al ., ; Konieczny and Helinski, ; Wickner et al ., ; Wickner et al ., ; Lu et al ., ; Konieczny and Liberek, ; Zhong et al ., ; Kongsuwan et al ., ; Wawrzycka et al ., ). The mutations that occurred in the trfA1 gene during evolution of pMS0506 in S. oneidensis were concentrated in the region coding for the predicted second helix within the TrfA1 N‐terminus region (Hughes et al ., ).…”