Interactions of DnaA Proteins from Distantly Related Bacteria with the Replication Origin of the Broad Host Range Plasmid RK2

Caspi, Ron; Helinski, Donald R.; Pacek, Marcin; Konieczny, Igor

doi:10.1074/jbc.m000552200

Cited by 35 publications

(25 citation statements)

References 41 publications

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“…The protocol and bacterial strain used for the purification of the E. coli β-clamp were kindly provided by D. Bastia (Medical University of South Carolina, Charleston, SC). E. coli proteins DnaA-His6, DnaB-His6, DnaC, and ClpX were purified as described (43,(47)(48)(49). All TrfA preparations used in the tests were N-terminally histidine-tagged 33-kDa versions of TrfA.…”

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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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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“…C-terminal His 6 -tagged DnaA proteins of E. coli, P. putida, and P. aeruginosa (21) and C-terminal His 6 -tagged DnaB proteins of E. coli, P. putida, and P. aeruginosa (20) were purified as described. The modified proteins have been found to behave similarly to the native E. coli proteins in several in vitro assays (28,29).…”

Section: Methodsmentioning

confidence: 99%

“…The simplicity of this system has allowed for a direct comparison of the mechanism for DNA replication initiation in different bacterial species (20,21).…”

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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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“…Surface Plasmon Resonance (SPR)-For the standard SPR analysis, a 64-bp 5Ј-biotinylated oligonucleotide (5Ј-AACGCCTGATTTTACGCG-AGTTTCCCACAGATGATGTG GACAAGCCTGGGGATAAGTGCCCT-GC-3Ј) and non-biotinylated complementary oligonucleotide were annealed and immobilized on to the Sensor Chip SA surface as described previously (1). SPR analysis was performed on a BiaCore 1000, by injecting 10 l of protein solutions in binding buffer (40 mM HEPES/KOH, pH 8.0, 40 mM potassium glutamate, 80 g/ml BSA, 4% sucrose, 4 mM dithiothreitol, 10 mM magnesium acetate, and 2 mM ATP) for 2 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

DnaA Box Sequences as the Site for Helicase Delivery during Plasmid RK2 Replication Initiation in Escherichia coli

Pacek¹,

Konopa

Konieczny³

2001

Journal of Biological Chemistry

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DnaA box sequences are a common motif present within the replication origin region of a diverse group of bacteria and prokaryotic extrachromosomal genetic elements. Although the origin opening caused by binding of the host DnaA protein has been shown to be critical for the loading of the DnaB helicase, to date there has been no direct evidence presented for the formation of the DnaB complex at the DnaA box site. For these studies, we used the replication origin of plasmid RK2 (oriV), containing a cluster of four DnaA boxes that bind DnaA proteins isolated from different bacterial species (Caspi, R., Helinski, D. R., Pacek, M., and Konieczny, I. (2000) J. Biol. Chem. 275, 18454 -18461). Size exclusion chromatography, surface plasmon resonance, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized ϳ200 base pairs upstream from the region of origin opening and a potential site for helicase entry. The DnaABC complex was formed on both double-stranded superhelical and linear RK2 templates. A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed. In addition, our experiments provide evidence for interaction between the plasmid initiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region. This interaction is facilitated via direct contact between TrfA and DnaB proteins.The initiation of DNA replication of prokaryotic and eukaryotic replicons requires delivering and loading of a helicase at the replication origin (2, 3). In addition to unwinding doublestranded DNA, the helicase has to be positioned on the separated DNA strands. Although there is considerable information of helicase delivery to the origin and loading on ssDNA, 1 the molecular mechanisms of these events are not fully understood.DnaA box sequences are present within replication origins of many different bacterial species (4 -9) and extrachromosomal prokaryotic genetic elements (10 -20). Originally, the DnaA binding site consensus sequences was determined for the Escherichia coli DnaA protein (21). It has been shown that the consequence of DnaA protein binding to a DnaA box sequence can result in various types of functions, depending on the bacterial system. Binding of DnaA protein to the DnaA box consensus sequence, localized within a promotor region, can stimulate or inhibit transcriptional activity, hence, the regulation of gene expression and/or stimulation of the initiation of DNA replication (22). However, in most cases, the binding of DnaA protein to specific DnaA boxes, localized within bacterial replication origins, is essential for the initiation of DNA replication. The binding of DnaA to DnaA boxes at the E. coli chromosomal replication origin results in destabilization of duplex DNA at the AϩT-rich region and an open complex formation (4, 23-26). The DnaB helicase (27), in the form of a DnaB/DnaC complex, is specifically loaded at the DnaA-induced open region of oriC (28 -30). Physical interactions b...

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Interactions of DnaA Proteins from Distantly Related Bacteria with the Replication Origin of the Broad Host Range Plasmid RK2

Cited by 35 publications

References 41 publications

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

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

DnaA Box Sequences as the Site for Helicase Delivery during Plasmid RK2 Replication Initiation in Escherichia coli

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