Conformation of a plasmid replication initiator protein affects its proteolysis by ClpXP system

Pierechod, Marcin; Nowak, Agnieszka; Saari, Anna; Purta, Elżbieta; Bujnicki, Janusz M.; Konieczny, Igor

doi:10.1002/pro.68

Cited by 20 publications

(51 citation statements)

References 68 publications

(92 reference statements)

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“…The clamp loader is responsible for β-clamp opening and loading it onto the primed DNA. The QLSLF motif in TrfA is located in the proximity of the WH domains responsible for the protein's interaction with DNA (43). It is worth noting that changing the QLSLF motif into ALSMA also results in a replicatively inactive TrfA (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…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%

“…Detailed information on the commercially available proteins, antibodies, and reagents used in this study is given in SI Materials and Methods. An in vitro replication assay (FII) was performed as described by Kittell and Helinski (50), ClpX-dependent activation of TrfA was performed according to the method of Pierechod et al (43), and ELISA analyses were performed as described previously (36), with details given in SI Materials and Methods. Detailed protocols for in vivo replication activity, isolation of nucleoprotein complexes by gel filtration, helicase activity assay (FI*), SPR, and GMSA analysis are given in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

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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: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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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“…QLSLF indicates the conserved amino acid sequence motif responsible for interaction with DNA polymerase III (33). DBD1 and DBD2 indicate DNA-binding (winged-helix) domains deduced from secondary structure comparisons (22,49). (B) The trfA gene locus of pBP136 and its mini-pBP136 derivative pMS0506.…”

Section: Methodsmentioning

confidence: 99%

“…TrfA-33 is the truncated version of TrfA-44 and is produced from a second translational start site in the trfA-44 reading frame. According to homology modeling, TrfA-33 shows structural similarity to Rep proteins from narrowhost-range plasmids such as the F plasmid and pPS10 (49). The N terminus of TrfA-44 is absent in the Rep proteins of those narrowhost-range plasmids (Fig.…”

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confidence: 99%

Roles of Long and Short Replication Initiation Proteins in the Fate of IncP-1 Plasmids

et al. 2012

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Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1␤ plasmid and its trfA1 frameshift variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, and even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2 or TrfA1 alone, again, generally elicited a higher PCN of an IncP1-␤ replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a 3-to 4-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts. P lasmids belonging to incompatibility group P-1 (IncP-1 group) are generally considered to have a broad host range owing to their ability to replicate in at least two classes within the phylum Proteobacteria (e.g., Alphaproteobacteria and Gammaproteobacteria). The majority of IncP-1 plasmids have been identified as agents that mediate transfer of beneficial traits between these phylogenetically distinct bacteria, such as antibiotic resistance, resistance to heavy metals, or catabolic activity of xenobiotic compounds (2, 51, 60). Since the worldwide spread of antibiotic resistance is a growing concern in human health care (40), it is important that we improve our understanding of the mechanisms by which IncP-1 plasmids are maintained in a broad range of hosts.Plasmid RK2, identified in Pseudomonas aeruginosa, has been studied as a model IncP-1 plasmid to gain understanding of basic plasmid functions (i.e., replication, stable inheritance, and conjugative transfer) and of the promiscuous nature of the IncP-1 group (32). It is known that the replication initiation protein (trans-acting factor A, TrfA) and the replication origin (oriV) are responsible for RK2 replication in a broad range of hosts (20, 52). RK2 encodes two replication initiation proteins (Rep proteins), 56). TrfA-33 is the truncated version of TrfA-44 and is produced ...

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Characterization of a newly discovered putative DNA replication initiator from Paenibacillus polymyxa phage phiBP

Halgasova

Javorova

Bocanova

et al. 2023

Microbiological Research

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Conformation of a plasmid replication initiator protein affects its proteolysis by ClpXP system

Cited by 20 publications

References 68 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

Roles of Long and Short Replication Initiation Proteins in the Fate of IncP-1 Plasmids

Characterization of a newly discovered putative DNA replication initiator from Paenibacillus polymyxa phage phiBP

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