theta replication of plasmid

This functional cross-talk between RNAI and tRNAs may contribute to plasmid copy number deregulation associated with amino-acid starvation in relA strains used for recombinant gene expression, one of the key factors is limiting yield of large-scale recombinant expression (69). Coordinated replication of both strands leads to the formation of bubbles in the early stages of replication, seen as the Greek letter under electron microscopy. These plasmids replicate from an internal origin of replication to form dimeric circles. Note that the name for these Reps is incidental so sharing a name is no indication of related structure or mode of action. Origin of replication or ori to refer to the cis -ori, and replicon to refer to basic or minimal replicons. In this case, the function of replication initiator function is provided by a different Rep (RepC). official website and that any information you provide is encrypted Plasmids have been used as convenient models for the study of molecular mechanisms of replication and DNA repair due to their small size, dispensability to the host, and easy manipulation. These are plasmid copy number regulatory systems that include two elements: a ctRNA and a transcriptional repressor protein. official website and that any information you provide is encrypted In theta mode, the two replication forks move away from each other. Mutational analysis of the specific priming signal essential for DNA replication of the broad host-range plasmid RSF1010. This is the first proof for the existence of thetareplicating plasmids in lactococci. Spacing is critically relevant so that the distance matches the helical periodicity of the DNA double helix, allowing recognition of specific DNA sequences (1). In theta plasmids, lagging-strand synthesis is discontinuous and coordinated with leading-strand synthesis. Such specificity is not possible when multiple primers are needed, as in the case of lagging-strand synthesis in the chromosome. mSystems. We show here that this replication (i) is dependent on a plasmid-encoded replication protein (Rep) but not on a DNA structure typical for origins of most Rep-dependent plasmids and (ii) is initiated by DNA polymerase I (PolI). pAM1 is a plasmid isolated from Enterococcus faecalis which replicates in Bacillus subtilis by a unidirectional theta mechanism. and on how initiation is regulated in these plasmids. Progress in nucleic acid research and molecular biology. 8600 Rockville Pike Theta replication of bacterial DNA | Download Scientific Diagram Both hairpins and forked structures recruit PriA, which is the first step in the replisome initiation complex. In some replicons duplex melting depends on transcription, while other replicons rely on plasmid-encoded trans-acting proteins (Reps); primers for leading-strand synthesis can be generated through processing of a transcript or in other replicons by the action of host- or plasmid-encoded primases. To perform this rolling-circle mode of replication, genetic material needs to be circular. . Finally, The two new double-stranded plasmids are synthesized from the original double-stranded plasmid. Similarly, in R6K plasmids, binding of its cognate iterons bends the DNA and generates a wrapped nucleoprotein structure (18). Rep binding of two alternative binding sites (Rep promoter and plasmid ori) involves changes in conformation and oligomerization status of the Rep protein. In the theta and strand-displacement plasmid replication types, the replication is initiated by RNA priming, while in the rolling-circle replication, replication is initiated by the cleavage of plasmid DNA strands to generate a free 3-OH end. In terC-containing derivatives, theta-form molecules with two invariant endpoints accumulate. The https:// ensures that you are connecting to the Reconsidering plasmid maintenance factors for computational plasmid design. DNA polymerase I (Pol I) contributes to plasmid replication in several ways. Opening of the DNA duplex is necessary for replisome assembly, which in theta-type plasmids can be DnaA-dependent or PriA-dependent. Understanding plasmid replication and maintenance has therefore significant practical implications for the clinic and for bioremediation. The replication process has been studied in detail for pAM1, although the Rep proteins (RepE for pAM1, RepF for pIP501, and RepS for pSM19035) are 97% identical for all three plasmids and the three plasmids share a replisome structure, suggesting they share mechanisms for replication initiation and termination. suggesting that RepB of pLP60 is a theta-replicating protein. Plasmids 101: Stringent Regulation of Replication - Addgene The Rep protein may stay bound to the ori after initiation of replication, blocking progression of the replisome synthesizing the lagging strand (42). Theta Plasmid Replication See this image and copyright information in PMC. doi: 10.1128/microbiolspec.PLAS-0029-2014. Plasmid In ColE1 and ColE1-like plasmids, Pol I can extend a primer to initiate leading-strand synthesis and open the DNA duplex; this process can expose a hairpin structure in the lagging-strand, known as single strand initiation (ssi) site or primosome assembly (pas) site, and/or generate a D-loop. Mechanisms of Theta Plasmid Replication in Enterobacteria and Implications for Adaptation to Its Host. In theta plasmids, inhibition of replication occurs at the initiation step and depends on three types of mechanisms: (a) antisense RNAs that hybridize to a complementary region of an essential RNA (countertranscribed RNAs, or ctRNAs) -dual mechanisms involving ctRNA and an additional protein repressor also occur- (b) Rep binding of iterons located in the Rep promoter, suppressing transcription; (c) steric hindrance between plasmids by interaction between Rep initiator proteins bound to different plasmids handcuffing them. Wu YC, Liu ST. A sequence that affects the copy number and stability of pSW200 and ColE1. This holoenzyme contains a core (with , a catalytic subunit, and , a 3'5 exonuclease subunit), a 2 processivity factor, and a DnaX complex ATPase that loads 2 onto DNA and recruits the Pol III core to the newly loaded 2 (9). 2022 Mar 22;9(1):92. doi: 10.1038/s41597-022-01179-8. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Replicons have at least one origin of replication, or. Plasmids are autonomously replicating pieces of DNA. Class A theta plasmids include R1, RK2, R6K, pSC101, pPS10, F, and P. All these plasmids depend on Rep proteins for replication initiation: RepA for R1, pSC101, pPS10 and P1; Trf1 for RK1; and for R6K. Rep protein is the dimer that is formed of the two monomers. DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number. In some replicons duplex melting depends on transcription, while other replicons rely on plasmid-encoded trans-acting proteins (Reps); primers for leading-strand synthesis can be generated through processing of a transcript or in other replicons by the action of host- or plasmid-encoded primases. Theta replication of the lactococcal plasmid pWVO2 - PubMed 2015 Feb;3(1):PLAS-0029-2014. Template switching has also . Giraldo R, Fernandez-Tresguerres ME. PDF Mechanisms of Theta Plasmid Replication Antisense RNA regulation of plasmid replication has been extensively reviewed elsewhere (55,56) and in a chapter of this book (57). By 2D agarose gel electrophoresis of replication intermediates, it was shown that pWVO2 replicates via a theta mechanism. eCollection 2023. Deletion of pcnB, the gene encoding PAPI, leads to increased cytoplasmic level of pRNAI cleavage intermediates and to a 5-to-10-fold (ColE1) and 2-fold (ColE2) decrease in plasmid copy number (61,62). This is the most common mode of DNA replication. Rannou O, Le Chatelier E, Larson MA, Nouri H, Dalmais B, Laughton C, Janniere L, Soultanas P. Functional interplay of DnaE polymerase, DnaG primase and DnaC helicase within a ternary complex, and primase to polymerase hand-off during lagging strand DNA replication in Bacillus subtilis. This is the first proof for the existence of thetareplicating plasmids in lactococci. This chapter focuses on replication of circular plasmids whose lagging-strand is synthesized discontinuously, a mechanism known as theta replication because replication intermediates have the shape of the Greek letter (theta). 2023 Feb 16;14:1112307. doi: 10.3389/fmicb.2023.1112307. The 2 micron plasmid: a selfish genetic element with an optimized 1998 Jun;62(2):434-64. doi: 10.1128/MMBR.62.2.434-464.1998. When the Rep protein has made a break in the DSO sequence, two ends will be formed in the DNA. DNA unwinds at the ori site from where the replication begins. Urh M, Wu J, Wu J, Forest K, Inman RB, Filutowicz M. Assemblies of replication initiator protein on symmetric and asymmetric DNA sequences depend on multiple protein oligomerization surfaces. Keywords: . The last section of the chapter reviews how plasmid copy number is sensed and how this feedback modulates the frequency of replication. The point where the RNA primer is extended (known as RNA/DNA switch) is considered the replication start point (reviewed in (27-29)). government site. Sakai H, Komano T. DNA replication of IncQ broad-host-range plasmids in gram-negative bacteria. Plasmids are circular or linear extrachromosomal replicons that are found in many microorganisms in the domains Bacteria, Archaea, and Eukaryota (Funnell and Phillips, 2004 ). Mechanisms of Theta Plasmid Replication in Enterobacteria and Point mutations at the loop portion of stem-loops are frequently tolerated, as mutations in the template DNA introduce complementary changes in sense and ctRNA at the same time, preserving base-pairing. However, loss of topoisomerase I and RNAse H activity do not increase plasmid copy number despite inducing increased R-loop formation because these activities are also required for plasmid replication initiation (particularly RNAse H). Rawlings DE, Tietze E. Comparative biology of IncQ and IncQ-like plasmids. II) The DNA duplex is melted through binding of RepC (possibly in concert with the RepA helicase), allowing the two ssi sites to form hairpins (ball and stick). Detection and characterization of a theta-replicating plasmid pLP60 BMC Microbiol. 2020 Nov;9(1):10.1128/ecosalplus.ESP-0026-2019. Given that target pre-primer and ctRNA sequence are complementary, higher order structures for both RNAs are mirror images of each other. In this case, RNAI is complementary to the 5 end of rep mRNA containing untranslated sequence. the contents by NLM or the National Institutes of Health. Kim JW, Bugata V, Corts-Corts G, Quevedo-Martnez G, Camps M. EcoSal Plus. An official website of the United States government. Strand-displacement replication depends on a specialized primase: RepB. Das N, Valjavec-Gratian M, Basuray AN, Fekete RA, Papp PP, Paulsson J, Chattoraj DK. Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication. I) Parental DNA duplex (solid, MeSH Learning more about the diversity of replication mechanisms present in plasmids can help understand the mechanisms that cells have available to replicate and repair their DNA. Tanaka K, Kino K, Taguchi Y, Miao DM, Honda Y, Sakai H, Komano T, Bagdasarian M. Functional difference between the two oppositely oriented priming signals essential for the initiation of the broad host-range plasmid RSF1010 DNA replication. sharing sensitive information, make sure youre on a federal Global Distribution and Diversity of Prevalent Sewage Water Plasmidomes. Plasmids are autonomously replicating pieces of DNA. K08 CA116429/CA/NCI NIH HHS/United States, R01 ES019625/ES/NIEHS NIH HHS/United States. Break-induced replication orchestrates resection-dependent template Malleret C, Zagorec M. Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low . An official website of the United States government. For their replication, plasmids encode only a few of the proteins. In the case of strand displacement, replication initiation is mediated by RepC, which (similar to class A theta . Front Microbiol. FOIA The theta mechanism is the most common form especially in Gram-negative bacteria like the proteobacteria. Replisome assembly is completed by loading of the Pol III holoenzyme (8). Low concentrations of RepA favor dissociation of Rep dimers into monomers, which are the only form that is active as initiator. 2015 Feb;3(1):PLAS-0035-2014. Replication and control of circular bacterial plasmids - PubMed Drolet M. Growth inhibition mediated by excess negative supercoiling: the interplay between transcription elongation, R-loop formation and DNA topology. In any case, ori acts as an enhancer, favoring the long-range activation of and oris by transfer of . Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Thus, in this case loading of DnaB is independent of DnaA. Note that continuous replication includes the lagging strand, which in this case does not involve synthesis of Okazaki primers (53). In this case the two regulatory elements are RNAIII and a Cop protein. Replication depends on transcription through the origin. Rep molecules are paired through zipping-up DNA-bound RepA monomers (78). In plasmid pSC101, RepA serves to stabilize DnaA binding to distant dnaA boxes, leading to strand melting (20). Class C and D theta plasmid replication is based on the evolution of more efficient ways to prime replication initiation. Controlled expression of two Rep proteins ( of R6K or RepA of ColE2) can produce a wide range of plasmid copy numbers per cell (between 1 and 250 copies), providing a convenient system for gene dosage optimization of recombinant proteins (2). Transcriptiondriven DNA replication of plasmid pAM1 in Bacillus Yavachev L, Ivanov I. Wu CA, Zechner EL, Marians KJ. This conformational change is mediated by the interaction of a sequence domain () in the pre-primer with another sequence domain further downstream (Y), making the pre-primer incompetent for R-loop formation. Ruiz-Mas JA, MachN C, Bordanaba-Ruiseco L, Espinosa M, Coll M, Del Solar G. Microbiol Spectr. PriA supports two distinct pathways for replication restart in UV-irradiated Escherichia coli cells. The cell also contains the proteins that help to initiate the replication. Elucidation of the structure of termination proteins (RTP and Tus) acting at specific termination sites has opened new avenues to perform detailed mechanistic analysis of this stage of replication . HHS Vulnerability Disclosure, Help Johnson A, O'Donnell M. Cellular DNA replicases: components and dynamics at the replication fork. The second element is a transcriptional repressor of P2, CopB. Since the structure resembles the Greek letter theta (), its name has been derived from it. Proceedings of the National Academy of Sciences of the United States of America. The ori site like theta-type could be located upstream of repB, which contains a short tandem repeats (sTR) and a long tandem repeats (lTR). To ensure autonomy from the host's replication and to facilitate copy number regulation, theta plasmids have unique mechanisms of replication initiation at the plasmid origin of replication ( ori ). Diaz-Lopez T, Lages-Gonzalo M, Serrano-Lopez A, Alfonso C, Rivas G, Diaz-Orejas R, Giraldo R. Structural changes in RepA, a plasmid replication initiator, upon binding to origin DNA. The Pol III holoenzyme extends off of the synthesized primer (Fig. official website and that any information you provide is encrypted Tight plasmid copy number regulation is essential because of the major and direct impact plasmid gene dosage has on gene expression. Frontiers in bioscience : a journal and virtual library. None of these processes require DNA breaks. Arch Microbiol. Structure of the ColE1 DNA molecule before segregation to daughter molecules. This review focuses on theta. Transcription-driven DNA replication of plasmid pAM1 in Gregg AV, McGlynn P, Jaktaji RP, Lloyd RG. In some cases, monomerization can be assisted by chaperones or by the allosteric effect of binding iterons at the ori (74-77). The pWVO2 minimal replicon is strongly related to that of several other lactococcal plasmid replicons. Replication starts at a cis site, designated ori, and proceeds by one of the two major DNA replication modes described for circular plasmids, rolling circle (RC) or theta (Thomas, 2000). Malmgren C, Engdahl HM, Romby P, Wagner EG. This autonomous replication initiation gives these plasmids a very broad range of operation (52). The study of specific signals involved in the termination of replication of theta-type plasmid replicons is a matter of growing interest. Clipboard, Search History, and several other advanced features are temporarily unavailable. I) Parental DNA duplex (solid black lines) depicting the two single-stranded replication initiation sites, ssiA (light gray box) and ssiB (dark gray box). are two types of bacterial DNA replication: the first is theta replication which takes place in circular DNA of bacteria such as E. coli. Plasmids contribute to adaptation of bacterial hosts to an ever-changing environment through mobilization and amplification of selected genes. Mechanisms of Theta Plasmid Replication in Enterobacteria and Drolet M, Broccoli S, Rallu F, Hraiky C, Fortin C, Masse E, Baaklini I. CopB expression is under the control of P1 but not P2. Takechi S, Itoh T. Initiation of unidirectional ColE2 DNA replication by a unique priming mechanism. Processing of plasmid DNA with ColE1-like replication origin. (b) The R6K plasmid, which has three origins of replication, only one of which has multiple iterons: (with 7 iterons); a second origin () features a single iteron; and a third origin () only has half an iteron. A different feedback mechanism that is known as steric hindrance or handcuffing was initially proposed for P1 and R6K plasmids (78,79) but could operate in more iteron-containing plasmids. Microbiol Spectr. The same reaction is used to re-form a circular plasmid after conjugational transfer. Plasmid replication is initiated by an interaction between a plasmid replication primer and the host DNA polymerase.