The Plasmid RK2 Initiation Protein Binds to the Origin of Replication as a Monomer

Toukdarian, Aresa; Helinski, Donald R.; Perri, Sílvia Helena Venturoli

doi:10.1074/jbc.271.12.7072

Cited by 41 publications

(61 citation statements)

References 47 publications

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“…E. coli DnaA, DnaB, DnaC, DnaG primase, DNA gyrase, polymerase III holoenzyme, and SSB proteins are required for RK2 replication in crude extracts (38,39) and in an in vitro replication system reconstituted with purified components. 3 The binding of the TrfA protein to the iterons at the minimal RK2 origin of replication has been characterized in detail (25,26,37,40); however, little is known about the nature of binding of the DnaA protein to oriV. We examined this binding using both the gel mobility shift assay and DNase I footprinting.…”

Section: Resultsmentioning

confidence: 99%

Role of TrfA and DnaA Proteins in Origin Opening during Initiation of DNA Replication of the Broad Host Range Plasmid RK2

Konieczny¹,

Doran

Helinski³

et al. 1997

Journal of Biological Chemistry

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125

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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.

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Section: Resultsmentioning

confidence: 99%

Role of TrfA and DnaA Proteins in Origin Opening during Initiation of DNA Replication of the Broad Host Range Plasmid RK2

Konieczny¹,

Doran

Helinski³

et al. 1997

Journal of Biological Chemistry

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125

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“…All TrfA preparations used in the tests were N-terminally histidine-tagged 33-kDa versions of TrfA. The wt TrfA, TrfA F138A, and TrfA ΔLF were purified according to the method of Toukdarian et al (33). E. coli DNA Pol III subunits were a kind gift from M. O'Donnell, The Rockefeller University, New York, NY.…”

Section: Methodsmentioning

confidence: 99%

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

Wawrzycka

Gross

Wasaznik

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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

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“…A previous study had shown that although wild type TrfA protein is primarily a dimer in solution, it was the monomeric form that was essential for replication initiation activity (26). Plasmid pGC1 served as the template for the construction of three specific N-terminal deletions and one point mutant as follows.…”

Section: Methodsmentioning

confidence: 99%

A Specific Region in the N Terminus of a Replication Initiation Protein of Plasmid RK2 Is Required for Recruitment of Pseudomonas aeruginosa DnaB Helicase to the Plasmid Origin

Zhong¹,

Helinski

Toukdarian

2003

Journal of Biological Chemistry

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Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21-32 yielded a mutant protein, TrfA-44⌬2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37-55 (TrfA-44⌬3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71-88 (TrfA-44⌬4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication.A critical step in the initiation of DNA replication is the recruitment, loading, and activation of the replicative helicase. Helicase activity is not only necessary for progression of the replication fork but, as studies with the Escherichia coli chromosomal origin oriC have revealed, the DnaB helicase makes essential contacts with other proteins in the replication complex. Recruitment of the helicase in E. coli requires association of the DnaB hexamer with the E. coli DnaC accessory protein (1-3). The DnaB-DnaC complex interacts with the host initiation protein, DnaA, bound to specific sequences (DnaA boxes) at oriC. This association results in the loading of the helicase onto unwound single-stranded DNA in the origin and the release of DnaC (4 -7). Once loaded onto the single-stranded DNA, DnaB interacts directly with DnaG to facilitate primase loading onto the single-stranded DNA at the replication fork (8,9). This interaction becomes the primary regulator of Okazaki fragment synthesis (10) and also ensures the proper placement of primers for leading strand synthesis (11). The subsequent association of DnaB with the Tau subunit of the DNA polymerase III holoenzyme results in rapid movement of the replication fork (12, 13).Studies on plasmids and phages that replicate in E. coli have revealed additional strategies for recruiting DnaB to a replication origin. The replication initiation...

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The Plasmid RK2 Initiation Protein Binds to the Origin of Replication as a Monomer

Cited by 41 publications

References 47 publications

Role of TrfA and DnaA Proteins in Origin Opening during Initiation of DNA Replication of the Broad Host Range Plasmid RK2

Role of TrfA and DnaA Proteins in Origin Opening during Initiation of DNA Replication of the Broad Host Range Plasmid RK2

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

A Specific Region in the N Terminus of a Replication Initiation Protein of Plasmid RK2 Is Required for Recruitment of Pseudomonas aeruginosa DnaB Helicase to the Plasmid Origin

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