title: 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.
authors.affiliation: Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822 Gdansk, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Wawrzycka
authors.affiliation: Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822 Gdansk, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Gross
authors.affiliation: Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822 Gdansk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Wasaznik
authors.affiliation: Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822 Gdansk, Poland igor.konieczny@biotech.ug.edu.pl.
authors.forename: Igor
authors.initials: I
authors.lastname: Konieczny
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D002942:Circular Dichroism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D053488:DnaB Helicases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009698:Nucleoproteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D018741:Replication Origin; D013698:Templates, Genetic; D000099272:Replisomes
chemical_list: D000251:Adenosine Triphosphatases; D004259:DNA-Directed DNA Polymerase; D053488:DnaB Helicases; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D050505:Mutant Proteins; D009698:Nucleoproteins; D021122:Protein Subunits; D000099272:Replisomes; C067405:TrfA protein, E coli
keywords: DNA replication initiation; Rep; plasmid RK2; polymerase III; β-clamp