Control of plasmid DNA replication. As discussed below, iterons not only are essential for replication but also are key elements for the control of plasmid replication (reviewed in references 51, 87, 155, and 223). Similar results have been obtained with the RepA protein of plasmid pSC101. Sites 1 and 2 share a core sequence (g/tTTAAA) that is an imperfect palindrome (101). Erauso G, Marsin S, Benbouzid-Rollet N, Baucher M F, Barbeyron T, Zivanovic Y, Prieur D, Fonterre P. Sequence of plasmid pGT5 from the archaeon. A model, similar to the one proposed by Novick for pT181 RC replication, has been recently proposed for termination of ssDNA conjugal transfer mediated by a dimer of the TraI protein of the IncP plasmid RP4 (240). pUC19 Vector map of pUC19 pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The mechanism of this arrest is unknown. Garca de Viedma D, Serrano-Lopez A, Diaz-Orejas R. Specific binding of the replication protein of plasmid pPS10 to direct and inverted repeats is mediated by an HTH motif. Copy number control in R1 is exerted at the level of RepA synthesis, which is modulated by the products of the copy number control genes, copB and copA (reviewed in references 224 and 309). The PcrA protein is essential for cell viability and for replication of plasmid pT181. Plasmid initiator proteins have drawn much interest, and the studies on these activators have been focused on the specific protein-protein and protein-DNA interactions involved in the formation of an active initiation complex. Lin J, Helinski D R. Analysis of mutations in. Analysis of the chirality of the phosphate involved in the cleavage reaction has shown that the nicking and closing reaction mediated by RepB is exerted through an even number of steps, suggesting that a covalent bond, albeit a transient one, between RepB and its target exists (202). DnaA binds to a dnaA box that is adjacent to the RepA-binding region, but this binding does not occur, or is very inefficient, in the absence of RepA (184, 233). 20C Description General description A versatile cloning vector for the expression of genes in mammalian cells. However, the enhancer favors the long-range activation of ori- and ori- by transfer of the initiator protein, and possibly other initiation factors, from ori- (199, 203, 204). See the text for details. The 3-OH end required for initiation of replication is provided by the site-specific nicking activity of the plasmid-encoded Rep protein on one of the parental plasmid strands. Individual plasmid copies are selected for replication at random from a pool that includes replicated and nonreplicated copies. Mutations affecting the CS-6 sequence of the pMV158-ssoA moderately increase the intracellular amount of plasmid ssDNA, although the effect is not as dramatic as that of the deletion of the entire ssoA (165, 167). What Is Origin Of Replication? - Researchtopics.quest The plasmid ori includes not only the specific sequences where the Rep and DnaA proteins interact but also an AT-rich region containing direct repeats, analogous to the 13-mers in oriC, where the DNA strands are melted. It is assumed that the RepA helicase binds to both DNA strands in the AT-rich region, close to the site of interaction of RepC. In: Thomas C M, editor. (ii) Although this is not a general feature, many plasmids encode a protein involved in the initiation of replication, usually termed Rep protein. There is only one essential module. The points at which theta-type replication terminates can be actively determined by molecular interactions at particular sequences. RepA protein, probably as a dimer, recognizes sequentially (albeit with different affinities) the cores of two partially palindromic sequences (Fig. Specific cleavage by RNase H of the preprimer (termed RNA II) annealed to DNA provides a 3 end that defines the starting point for leading-strand synthesis. DNA Pol III is required for elongation of plasmid DNA replication. Some common ones you might see include ColE1, pMB1 (which comes in a few slightly different but well known derivatives), pSC101, R6K, and 15A. Bacteria tend to maintain fewer copies of plasmids if they contain large inserts or genes that create a toxic product. Zhao A C, Khan S A. Conservation of the nic locus and divergence in the bind region can also be observed in the plasmids of the pMV158 family. This is achieved by plasmid-encoded control elements that regulate the initiation of the replication step. Konieczny I, Doran K S, Helinski D R, Blasina A. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions. Bethesda, MD 20894, Web Policies In the ssoA of the plasmid group represented by pMV158, the central conserved sequence, termed CS-6, is 5-TAGCGt/a-3 (65). However, once the characteristic copy number is reached, keeping the average copy number in the population requires adjustments to fluctuations in this value in individual cells. I. 8600 Rockville Pike In addition to the host RNAP, DNA Pol I is involved in lagging-strand replication both in initiation and in termination (76, 166). Centro de Investigaciones Biolgicas, CSIC, E-28006 Madrid, Spain, {"type":"entrez-protein","attrs":{"text":"RSF10110","term_id":"1534199429"}}, {"type":"entrez-nucleotide","attrs":{"text":"X58896","term_id":"45872"}}, {"type":"entrez-nucleotide","attrs":{"text":"Y10829","term_id":"1808661"}}, {"type":"entrez-nucleotide","attrs":{"text":"L30112","term_id":"463167"}}, {"type":"entrez-nucleotide","attrs":{"text":"X86092","term_id":"769802"}}, {"type":"entrez-nucleotide","attrs":{"text":"M31727","term_id":"148533"}}, {"type":"entrez-nucleotide","attrs":{"text":"K00828","term_id":"150939"}}, {"type":"entrez-nucleotide","attrs":{"text":"Z11775","term_id":"43899"}}, {"type":"entrez-nucleotide","attrs":{"text":"Y00547","term_id":"45794"}}, {"type":"entrez-nucleotide","attrs":{"text":"M65025","term_id":"151841"}}. Eguchi Y, Itoh T, Tomizawa J. Antisense RNA. Solid lines, parental DNA; broken lines, newly synthesized DNA. Gel filtration studies have shown that the apparent molecular weight of RepC is consistent with dimer formation (257). Khatri G S, MacAllister T, Sista P R, Bastia D. The replication terminator protein of. Concerning lagging-strand synthesis, only host factors are thought to be required in plasmids replicating by the RC and theta modes. Replication starts when these origins are exposed as single-stranded regions. A detailed study of the site of RepA interaction revealed two RepA-binding sites: a preferential RepA site, termed site 1 (5-CAGTTAAATG-3), which is adjacent to the dnaA box, and a related RepA binding sequence, site 2 (5-TGTTTAAAAG-3), for which the protein has a lower affinity. Inhibition under overexpression conditions was explained by assuming that elevated concentrations of RepA would promote its dimerization and that the RepA dimers would hinder the interaction of the active RepA monomeric forms with the iterons of the origin (134). A quantitative model for control of plasmid NR1 replication in the bacterial cell division cycle. A second element involved in copy number control is a small countertranscribed RNA, RNA II, which is complementary to a region of the cop-rep mRNA between genes copG and repB. Thompson J D, Higgins D G, Gibson T J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. as a modification of an existing group of cloning plasmids. Evidence of two levels of control of P1. As stated above, replication from the ori of R6K requires protein (96, 160, 272, 278). However, complete inactivation of CopA leads to uncontrolled (runaway) replication (reviewed in references 224 and 226). However, mechanisms that counterselect newly replicated molecules exist, e.g., hemimethylation and supercoiling (3, 224). The newly replicated, hemimethylated DNA is sequestered in the cell membrane, thus preventing reinitiation. Brendler T, Abeles A, Austin S J. The second regulatory element in R1, the CopB protein, plays an auxiliary role under normal circumstances. The initiator promotes the initiation of replication from three origins of replication: , , and (reviewed in reference 87). Although plasmid copy number may vary in different bacteria, within a given host, and under fixed growth conditions, any particular plasmid has a characteristic copy number. Theta-type replication is, in most cases, unidirectional. Formation of this initial contact (or kissing complex) is the rate-limiting step of the interaction and leads to the full annealing of the antisense RNA I with the preprimer RNA II, in a process that starts at the 5 end of the RNA I. Little information is available on the fourth plasmid family, and newly described plasmids from Pyrococcus abyssi and from extreme halophiles seem to belong to one of these families or to fall within a different, less well characterized, fifth family (83, 127). del Solar G, Moscoso M, Espinosa M. Rolling circle-replicating plasmids from gram-positive and -negative bacteria: a wall falls. Vocke C, Bastia D. DNA-protein interaction at the origin of DNA replication of the plasmid pSC101. These interactions increase, but are not strictly required for, the opening of the strands. The iteron-containing origin (oriV) and motifs found in the replication initiator protein (RepA) are depicted. These plasmids will also carry functions needed to be mobilized or mob genes. [2] This independence may account for the broad-host-range character of the IncQ replicons. The current model for the control of replication of pT181 (references 227 and 228 and references therein) proposes that synthesis of the Rep initiator is limited by two small ctRNAs (having the same 5 end but different 3 ends), which are complementary to the untranslated 5 end of the rep mRNA (44, 119, 227, 229). DNA Pol I synthesizes about 400 nucleotides of the leading strand, exposing, on the displaced strand, a primosome assembly site (pas). Frey J, Bagdasarian M. The molecular biology of IncQ plasmids. The origins of replication can also contain sites for factors (e.g., the integration host factor, IHF, or the factor for inversion stimulation, FIS) that play an architectural role. Ozaki E, Yashubara H, Masamune Y. Purification of pKYM-encoded RepK, a protein required for the initiation of plasmid replication. Kelley W L, Patel I, Bastia D. Structural and functional analysis of a replication enhancer: separation of the enhancer activity from origin function by mutational dissection of the replication origin gamma of plasmid R6K. After completion of leading-strand synthesis, the Rep protein is inactivated and plasmid ssDNA intermediates are generated. Generally, control of replication is referred to as "relaxed" or "stringent" depending on whether theoriis positively regulated by RNA or proteins, respectively. The feature that better defines plasmids is that they replicate in an autonomous and self-controlled way. A mutation at the 3 end of the trfA gene (affecting the two versions of the protein) modifies the host range of RK2 without altering the binding of the protein to DNA (45, 175; also see reference 241). A computer model that adjust to this hypothesis has been developed (321). The minimal ori region includes three identical 20-bp iterons plus a 174-bp region that contains a GC-rich stretch (28 bp) and an AT-rich segment (31 bp). Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. Wojtkowiak D, Georgopoulos C, Zylicz M. Isolation and characterization of ClpX, a new ATP-dependent specificity component of the Clp protease of. Proposal for the domains Archaea, Bacteria and Eucarya. Pouwels P H, van Luijk N, Leer R J, Posno M. Control of replication of the. Womble D D, Rownd R H. Regulation of mini-F plasmid DNA replication. It has recently been suggested that in particular plasmids in which initiators are limiting, initiator titration and initiator pairing (handcuffing) could be working together (51). In: Molineux I, Kohiyama M, editors. **F1 is a phage-derived ori that allows for the replication and packaging of ssDNA into phage particles. Plasmids are double-stranded, self-replicating DNA segments with a few kilobases that are often found in gram-negative and gram-positive bacterial strains, as well as various fungi, including unicellular yeasts. For instance, removal of one of the seven iterons from ori- of plasmid R6K has no effect but deletion of two reduces the efficiency of replication and deletion of three or more abolishes plasmid replication (160). Replication of this displaced strand is initiated at the exposed ssi origin. A mechanism of formation of a persistent hybrid between elongating RNA and template DNA. Binding of RepA dimers to sites 1 and 2 would generate a small DNA loop, held together by protein-protein interactions. An active ori- requires the binding sites for , DnaA, and IHF proteins in the correct geometrical alignment (147). Based on amino acid sequence alignments of multiple Rep proteins from theta-replicating plasmids, it is possible to construct phylogenetic trees like the one depicted in Fig. Under steady-state conditions, CopB is present at saturating concentrations, fully repressing transcription from P2 to a basal level. The origin of replication determines the vector copy number, which could typically be in the range of 25-50 copies/cell if the expression vector is derived from the low-copy-number plasmid pBR322, or between 150 and 200 copies/cell if derived from the high-copy-number plasmid pUC. Why, you ask? Need something clarified? Nordstrm K, Ingram L C, Lundbck A. Mutations in R factors of. Plasmids with phage-derived ori's are referred to as phagemids. This suggests that the ssDNAdsDNA conversion requires unpaired sequences within the secondary structures that constitute the sso (65, 68, 109). Studies on the control of plasmid replication have revealed the role played by small antisense RNAs in the processing of RNAs or in the specific inhibition of Rep-protein synthesis at the transcriptional or translational level. Scherzinger E, Bagdasarian M M, Scholz P, Lurz R, Rckert B, Bagdasarian M. Replication of the broad host-range plasmid RSF1010: requirement for three plasmid encoded proteins. The data for P1 indicate that the concentration of the auxiliary iterons rather than their relative arrangements is the deterministic factor for replication control (51). In the plasmids of the pMV158 family, the higher degree of homology found at the N-terminal region than at the C terminus of their Rep proteins suggested that their conserved N-terminal moiety would be involved in nicking activities whereas the C termini would be involved in specific dso recognition (69). However, under conditions in which the copy number drops or is low because of the early stages of plasmid establishment, the CopB-mediated repression is not efficient. Seufert W, Messer W. DnaA protein binding to the plasmid origin region can substitute for primosome assembly during replication of pBR322 in vitro. Bird R E, Tomizawa J. Ribonucleotide-deoxyribonucleotide linkages at the origin of DNA replication of colicin E1 plasmid. Plasmid - Wikipedia What is the origin of replication in DNA replication? The ability of some plasmids to pass across the so-called genetic barriers among different living organisms has posed questions about general mechanisms governing replication and about the communication between plasmid replication components and the host machinery involved in DNA replication. Masai H, Kaziro Y, Arai K-I. However, and unlike R1, the CopR-inhibited rep promoter is not fully repressed, a situation resembling pMV158. DNA deformations at the origin may provide an appropriate configuration for the initiation event: cruciform extrusion and nicking by the initiator for RC-replicating plasmids versus assembly of the initiation complex and synthesis of an RNA primer for the other replicons. Whereas IR-II is conserved among plasmids of the pT181 family, IR-III is not, suggesting that the origin specificity is provided by IR-III. These considerations are especially useful to keep in mind if you are planning to purify your plasmid DNA: Have any questions? Data obtained with plasmid R1 indicate that maturation of newly replicated DNA molecules is a slow process, which prevents rapid reutilization of the last replicated molecules (221). Iterons can be adjacent or separated by intervening sequences. The model was based on the ability of Rep proteins to bind to two iterons simultaneously (in both P1 and R6K) (204). Koepsel R R, Khan S A. Static and initiator protein-enhanced bending of DNA at a replication origin. Analogous genetic structures in the control-of-replication region have been found in plasmids of the pMV158 family (69). However, in this area there is a lack of application of molecular biology techniques, which may result in a biased identification and characterization of plasmids isolated from new environments or from environments which have not been subjected to extreme selective pressures. This small dimeric and basic protein represses transcription of repA from a promoter, P2, which is located downstream of copB (Fig. This region also contains the origin of replication, as defined by EM analysis of replication intermediates obtained in vivo (59) and in vitro (266). Accessibility Pabo C O, Sauer R T. Protein-DNA recognition. The heptamer repeats containing the methylation sites in the origin of replication of plasmid P1 constitute the target of the host protein SeqA, involved in sequestering hemimethylated oriC into the bacterial membrane (37). The nic region contains inverted repeats able to generate one or two hairpin structures (65, 216, 254). The replication origin of a repABC plasmid - PubMed For plasmids of the pT181 family, studies with hybrid initiator proteins have demonstrated that a stretch of 6 amino acids, located at the C terminus of the Rep proteins, is sufficient to confer dso specificity, i.e., to interact with the nonconserved bind region of the cognate dso (73, 311). Birch P, Khan S. Replication of single-stranded plasmid pT181 DNA. Daz R, Nordstrm K, Staudenbauer W L. Plasmid R1 DNA replication dependent on protein synthesis in cell-free extracts of, Daz R, Staudenbauer W L. Origin and direction of mini-R1 plasmid DNA replication in cell extracts of. Furthermore, two other residues (Glu-142 and Glu-210) are also required for the catalytic activity (217). Some steps in these directions have recently been taken for the RepA protein of plasmid P1 (50). Article Series: E.coli Plasmid Origins of Replication: The Origin What is the ColE1 origin? - Studybuff.com Michelle R. McGehee, in Molecular Biology (Third Edition), 2019 2.1 Plasmid Families and Incompatibility Two different plasmids that belong to the same family cannot co-exist in the same cell. The development of a system in which DNA strand discontinuities can be mapped with nucleotide resolution in vivo has allowed the identification of the nick sites of other plasmids of the pMV158 family, namely, pE194, and pFX2 (106, 328). The streptococcal plasmid pMV158 has the interesting feature of bearing both ssoA and ssoU (68, 171, 250, 306). In pMV158, the pT181 model for termination of replication does not seem to be applicable, since RepB does not remain covalently bound to DNA (200). At an early stage, leading-strand synthesis proceeds in the absence of lagging-strand synthesis (297, 298). Priebe S D, Lacks S A. Kato J, Nishimura Y, Imamura R, Niki H, Hiraga S, Suzuki H. Gene organization in a region containing a new gene involved in chromosome partitioning in. Sista P R, Mukherjee S, Patel P, Khatri G S, Bastia D. A host-encoded DNA-binding protein promotes termination of plasmid replication at a sequence-specific replication terminus. Khan S A, Carleton S M, Novick R P. Replication of plasmid pT181 in vitro: requirement for a plasmid-encoded product. The footprints generated on supercoiled DNA by the RepC/C homodimer are different from those formed by the RepC/C* heterodimer, and whereas RepC/C was able to enhance cruciform extrusion at the dso, RepC/C* was not (140). These host-encoded proteins favor a topological proximity between different ori regions or even between different origins present in the same plasmid (as in plasmid R6K [see below]). This harbors the functions for plasmid replication and includes the dso, the rep gene, and the plasmid elements involved in replication control. Patel I, Bastia D. A replication initiator protein enhances the rate of hybrid formation between a silencer RNA and an activator RNA. Bruand C, Ehrlich S D, Jannire L. Primosome assembly site in, Bussiere D E, Bastia D, White S. Crystal structure of the replication terminator protein from. The function of the middle patch is less clear, but it may contribute to a proper conformation of the RepA-binding site. The DNA of the oriR region could be bent to facilitate the topological proximity of sites 1 and 2, which are disposed on the same face of DNA double helix. A single DnaA box is sufficient for initiation from the P1 plasmid origin. In these plasmids, the distance between the conserved nick site and the direct repeats (the nonconserved bind locus) ranges between 14 and 95 nucleotides. Daz A, Lacks S A, Lpez P. Multiple roles for DNA polymerase I in establishment and replication of the promiscuous plasmid pLS1. Roles of X174 type primase-dependent priming in initiation of lagging and leading strand synthesis of DNA replication. Initiation of pSC101 replication requires, in addition to RepA (308), the DnaA host replication initiator (113), and IHF proteins (91, 283). Minden J S, Marians K J. Replication of pBR322 DNA in vitro with purified proteins. The replication intermediates can also be monitored by one- or two-dimensional electrophoresis. Garca de Viedma D, Giraldo R, Rivas G, Fernandez-Tresguerres E, Diaz-Orejas R. A leucine zipper motif determines different functions in a DNA replication protein. In pMV158, in vitro RepB-dependent cleavage of the nick sequence absolutely requires ssDNA as the substrate (200, 201) and the hairpin corresponding to the nic region has been shown to extrude on supercoiled plasmid DNA (65, 254). In some cases, these proteins also participate directly in the generation of the primer. Most replication origins that are licensed during G1 phase never fire and remain dormant throughout S phase because their licensing factors are displaced by a passing fork from a more active origin [217].If replication slows or stalls, firing of these dormant origins can potentially act as a backup in order to complete replication (Fig. Replication by the RC mechanism has to be unidirectional, and it is considered to be an asymmetric process because synthesis of the leading strand and synthesis of the lagging strand are uncoupled (reviewed in references 69, 84, 108, 150, 150a, and 228). Also, let us know if you have any special requests for future Plasmids 101 series topics and stay tuned for the next installment. Ortega S, Lanka E, Daz R. The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA. In principle, the probability of Rep-mediated iteron pairing greatly increases in these replicons, since it can occur in cis by DNA looping and in various combinations in trans. The replicons of plasmids are generally different from the those used to replicate the host's chromosomal DNA, but they still rely on the host machinery to make additional copies. Origins of replication and related regions of plasmids replicating by the RC mode, as exemplified by plasmids pMV158 (A) and pT181 (B). How can I find a plasmid's origin of replication? | ResearchGate In addition, the presence of bulged nucleotides in the CopA stem seems to result in a structural destabilization that plays a protective role against degradation by RNase III (125). Concerning their genetic structure, plasmids have an essential region which contains the genes or loci involved in replication and its control. Carleton S M, Projan S J, Highlander S K, Moghazeh S M, Novick R P. Control of pT181 replication. Thomas C D, Nikiforov T T, Connolly B A, Shaw W V. Determination of sequence specificity between a plasmid replication initiator protein and the origin of replication. Replication from this origin requires the synthesis of an activator RNA that starts within the seven iterons of the origin and is silenced by the antisense RNA. Trawick J D, Kline C. A two-stage molecular model for control of mini-F replication. Since overproduction of host DnaA protein can reverse inhibition by excess of RepA (133), an alternative explanation to understand inhibition by excess of initiation proteins involves titration of host replication factors. The RNA I is synthesized from a constitutive promoter (therefore its level is proportional to the plasmid copy number) and is unstable. Broad host range plasmids | FEMS Microbiology Letters - Oxford Academic We are indebted to Deepak Bastia, Dhruba Chattoraj, Don Helinski, and Martine Couturier for critical reading of the manuscript and many useful comments and to Saleem Khan for many interesting discussions. . The study of specific signals involved in the termination of replication of theta-type plasmid replicons is a matter of growing interest. Interactions of plasmid initiator proteins with host replication factors have been reported in different systems: (i) the DnaJ protein interacts with the initiation protein of plasmid P1 (312a) and with other chaperones, promoting the efficient binding of this initiator to the origin of replication; (ii) the DnaA protein requires a functional interaction with the RepA protein of plasmid R1 to enter the DnaA box present in the origin of replication (184) (this protein interaction seems to be sufficient to promote DNA replication in the absence of the DnaA box [233]); and (iii) most interestingly, the DnaA, DnaB, and DnaG proteins of the host interact with the protein of plasmid R6K (16a, 258a) (mutations in the protein that disrupt the interaction with the DnaA protein are defective in R6K replication [16a]; the specific regions of DnaB and proteins involved in their interaction have been defined [258a]). Addgene Senior Scientist Marcy Patrick says researchers can ask themselves a few questions to get started: In other words, the best choice ofori depends on how many plasmid copies you want to maintain, which host or hosts you intend to use, and whether or not you need to consider your plasmid's compatibility with one or more other plasmids. Mukherjee S, Patel I, Bastia D. Conformational changes in a replication origin induced by an initiator protein. Interactions between the plasmid-encoded proteins RepC and RepA are indicated. Staudenbauer W L. Structure and replication of the colicin E1 plasmid. These sequences are used to initiate synthesis of the complementary strand, which converts the ssDNA templates into double-stranded supercoiled circles. The activation of ori-, unlike ori- and ori-, does not require DnaA protein (147). Dellis S, Filutowicz M. Integration host factor of. However, genetic evidence has shown that, at least for RepB of pMV158, the putative shorter RepB protein is not sufficient to conduct replication in vivo (70). Identification of a common fold in the replication terminator protein suggests a possible model for DNA binding. However, since the synthesis of RepU is autoregulated (207), a more complex control level may have to be considered. Plasmids 101: The Promoter Region - Let's Go! - Addgene Wickner S, Hoskins J, McKenney K. Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA. The initiator protein RepB acts on two distant DNA regions. This leaves a gap that is filled later in the replication cycle (186). Nordstrm K. Control of plasmid replication.

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