Structures of pMV158 replication initiator RepB with and without DNA reveal a flexible dual-function protein

DNA replication is essential to all living organisms as it ensures the fidelity of genetic material for the next generation of dividing cells. One of the simplest replication initiation mechanisms is the rolling circle replication. In the streptococcal plasmid pMV158, which confers antibiotic resistance to tetracycline, replication initiation is catalysed by RepB protein. The RepB N-terminal domain or origin binding domain binds to the recognition sequence (bind locus) of the double-strand origin of replication and cleaves one DNA strand at a specific site within the nic locus. Using biochemical and crystallographic analyses, here we show how the origin binding domain recognises and binds to the bind locus using structural elements removed from the active site, namely the recognition α helix, and a β-strand that organises upon binding. A new hexameric structure of full-length RepB that highlights the great flexibility of this protein is presented, which could account for its ability to perform different tasks, namely bind to two distinct loci and cleave one strand of DNA at the plasmid origin.© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research

Opposing effects of DNA on proteolysis of a replication initiator

DNA replication initiation proteins (Reps) are subjected to degradation by cellular proteases. We investigated how the formation of nucleoprotein complex, involving Rep and a protease, affects Rep degradation. All known Escherichia coli AAA+ cytosolic proteases and the replication initiation protein TrfA of the broad-host-range plasmid RK2 were used. Our results revealed that DNA influences the degradation process and that the observed effects are opposite and protease specific. In the case of ClpXP and ClpYQ proteases, DNA abolishes proteolysis, while in the case of ClpAP and Lon proteases it stimulates the process. ClpX and ClpY cannot interact with DNA-bound TrfA, while the ClpAP and Lon activities are enhanced by the formation of nucleoprotein complexes involving both the protease and TrfA. Lon has to interact with TrfA before contacting DNA, or this interaction can occur with TrfA already bound to DNA. The TrfA degradation by Lon can be carried out only on DNA. The absence of Lon results with higher stability of TrfA in the cell

Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA

15 p.-6 fig.An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA-protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin.National Science Centre [2012/04/A/NZ1/00048 to I.K.;2017/26/D/NZ1/00239 to K.W.]; Foundation for Polish Science [TEAM, POIR.04.04.00-00-5C75/17-00 to I.K.]; International Institute of Molecular and Cell Biology in Warsaw (to J.M.B.); Ministerio de Economía,Industria y Competitividad (MINECO/AEI) [BIO2012-30852, RTI2018-094549-B-I00 to R.G.]. Funding for open access charge: Foundation for Polish Science [TEAM,POIR.04.04.00-00-5C75/17-00].Peer reviewe

Broad-host range plasmids

Broad-host-range plasmids are model systems for exploring replication mechanisms in diverse bacterial species and can extend our understanding of universal rules guiding DNA metabolism. Despite extensive work carried out in many laboratories, several critical aspects of the plasmid DNA metabolism still remain unclear. This review is restricted to specification of the molecular events during theta replication initiation of the iteron containing broad-host-range plasmids. The discussion concerns RC (rolling circle) broad-host-range plasmids replicating via sigma mode and the well biochemically characterized initiation of replication at Escherichia coli oriC. Recent extensive biochemical investigations of replication initiation of narrow-host-range plasmids: pSC101, R1, P1, F, R6K, and broad-host- range plasmids RSF1010 and RK2 allow us to discuss the specificity of DNA replication initiation at broad-host- range origins

Strategies for helicase recruitment and loading in bacteria

DNA replication initiation in prokaryotes and eukaryotes requires the recruitment and loading of a helicase at the replication origin. To subsequently unwind the double-stranded DNA, the helicase must be properly positioned on the separated DNA strands. Several studies have revealed similarities and differences in the mechanisms used by different autonomously replicating DNA elements (replicons) for recruitment and activation of the appropriate helicase. Of particular interest are plasmid replicons that are adapted for replication in diverse bacterial hosts and are therefore intriguingly able to exploit the helicases of distantly related bacterial species. The different molecular mechanisms by which replicons recruit and load helicases are only just beginning to be understood

Radiographic Assessment of Tibiofibular Syndesmosis Injury with Different Durations and Types of Fixation

Introduction: There is no consensus among orthopedic surgeons on the number of cortical layers (tricortical or quadricortical fixation) involved or the duration of syndesmotic fixation after a tibiofibular syndesmosis (TFSD)-injury treatment. The purpose of this study was to assess radiographic parameters following the treatment of TFSD injuries, with various time-windows of syndesmotic screw removal and numbers of cortical layers involved. Materials and Methods: Fifty-five patients, aged from 25 to 75 years, were included in the study. The follow-up period ranged from 2 years to 4 years and 2 months. The patients were subdivided into groups based on the duration of the syndesmotic fixation (8–15 weeks—19 patients or 16–22 weeks—36 patients) and the number of cortices involved (tricortical—17 patients or quadricortical fixation—38 patients). Results: The quadricortical fixation group showed a significant development of ankle joint arthritis and subtalar joint arthritis at the final follow-up. The mean medial clear space was 2.84 mm in the tricortical fixation group and 3.5 mm in the quadricortical fixation group (p = 0.005). Both groups, with different screw removal times showed significant development of posttraumatic arthritis. A comparison of the two groups (with different time-windows of the screw removal) revealed a significant difference only in terms of the postoperative tibiofibular (TF) overlap and the observed rates of talonavicular arthritis at the final follow-up. Discussion: We found that the duration of the screw fixation had no effect on most of the evaluated radiographic parameters. Only the postoperative TF overlap was lower in the 8–15-week fixation group, and the proportion of patients with talonavicular joint arthritis at the final follow-up was higher in the 16–22-week fixation group. In addition, the number of cortices involved in the screw fixation had no effect on the radiographic outcomes in our patients, apart from the differences in one parameter—the medial clear space—at the final follow-up. Conclusion: We achieved similar radiographic results irrespective of the duration of the screw fixation and the number of cortices involved. All study subgroups showed the development of adjacent-joint arthritis following treatment. Considering the results of our study, the economic and medical aspects of treatment, and the possibility of a faster recovery, the most optimal solution seems to be the use of a tricortical fixation, with the screws being removed after 8–15 weeks

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

Proteins from the Rep family of DNA replication initiators exist mainly as dimers, but only monomers can initiate DNA replication by interaction with the replication origin (ori). In this study, we investigated both the activation (monomerization) and the degradation of the broad-host-range plasmid RK2 replication initiation protein TrfA, which we found to be a member of a class of DNA replication initiators containing winged helix (WH) domains. Our in vivo and in vitro experiments demonstrated that the ClpX-dependent activation of TrfA leading to replicationally active protein monomers and mutations affecting TrfA dimer formation, result in the inhibition of TrfA protein degradation by the ClpXP proteolytic system. These data revealed that the TrfA monomers and dimers are degraded at substantially different rates. Our data also show that the plasmid replication initiator activity and stability in E. coli cells are affected by ClpXP system only when the protein sustains dimeric form