Novick R P, Iordanescu S, Projan S J, Kornblum J, Edelman I. pT181 plasmid replication is regulated by a countertranscript-driven transcriptional attenuator. The two loci can be contiguous (pT181 family) or can be separated by up to about 100 bp (pMV158 family). The nucleotide sequence surrounding the replication terminus of R6K. Broad host range plasmids | FEMS Microbiology Letters - Oxford Academic Acebo P, Alda M T, Espinosa M, del Solar G. Isolation and characterization of pLS1 plasmid mutants with increased copy numbers. Molecular analysis of plasmid replication and its control occurring in bacterial populations growing under natural conditions should be considered. A quantitative model for control of plasmid NR1 replication in the bacterial cell division cycle. The Rep-DNA complex, in combination with DnaA, facilitates the transfer of the DnaB-DnaC complex to the origin and the opening of the strands in the AT-rich region. It is interesting that the amount of RepC protein of pT181 has been estimated to be one dimer molecule per plasmid replication event (16). The different ways in which antisense RNAs modulate the control of replication are well established. Kramer M G, Khan S A, Espinosa M. Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement of DNA polymerase I for lagging strand initiation. Although cross-activities of DNA gyrase in decatenation and of Topo IV in supporting fork progression can be detected, in vivo and in vitro data confirm the specialized role of Topo IV in unlinking daughter replicons (117, 245, 246, 327). Konieczny I, Doran K S, Helinski D R, Blasina A. These direct repeats seem to be essential for plasmid replication in vivo but not for in vitro relaxation of supercoiled DNA mediated by the plasmid Rep protein (201). Abeles A L, Reaves L D, Youngre-Grimes B, Austin S J. Under steady-state conditions, CopB is present at saturating concentrations, fully repressing transcription from P2 to a basal level. PSF-CMV-SV40 ORI SBFI - SV40 ORIGIN PLASMID - MilliporeSigma These events determine the unidirectional pattern of ColE1 replication. The ClpX chaperone yields monomers of the plasmid initiation protein, TrfA, which is the form that is active in binding to the five 17-bp iterons of the origin (161b). In the RC-replicating plasmid pMV158, a ctRNA has been proposed to inhibit the translation of the repB gene by direct interaction with the initiation of translation sequences present in the repB mRNA (67, 71, 72). A RepA initiator (hatched oval) dimer binds with high affinity to site 1, and then, in a second binding event, a different RepA dimer would bind with lower affinity to the distal site 2 sequence. This belief is probably wrong. In: Thomas C M, editor. The use of such alignments has allowed us the identification of Rep protein motifs, involved in protein-protein interaction (leucine zipper [LZ]) and in DNA binding (helix-turn-helix [HTH]) (93, 94, 103). A plasmid is a DNA molecule, other than the bacterial chromosomes that has the ability of replication and transmission independently. Some of the replicons containing iterons in the origin also contain iterons outside this region, which express incompatibility and further reduce the rate of replication (237). It also codes for immunity from this product with the imm gene. As the number of copies increases, Rep molecules bound to the iterons of one origin begin to interact with similar complexes generated on other origins. 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. In fact, the analyses of copy number control circuits of ColE1 led to the identification of antisense RNAs, a milestone discovery in molecular biology. Lagging-strand synthesis, initiated at the pas site, extends toward the promoter of RNA II but is arrested at a site (terH) 17 bp upstream of the leading-strand initiation site (57). The pPS10 replicon also contains the repA gene, encoding the RepA initiator protein (shadowed ovals). 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. 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. According to this model, it is the iteron concentration, rather than the level of Rep expression, that determines the rate of replication. The origin of ColE1 replication spans a region of about 1 kb that includes (i) sequences promoting the synthesis of RNA II, the primer of the leading strand (298, 299); (ii) sequences that allow a stable hybridization of RNA II to DNA (139, 189); (iii) sequences that favor specific processing of this coupled complex by RNase H, which generates the 3 end needed to prime leading-strand synthesis (122, 139); (iv) a primosome assembly site (pas or ssiA) that allows the loading of the DnaB helicase and DnaG primase to initiate the discontinuous priming of the lagging strand (28, 189, 220) (a dnaA box that is close to pas can be used as a DnaA-dependent DnaB-DnaC assembly site [269, 270]); and (v) a sequence for termination of lagging-strand synthesis, terH, which determines unidirectional replication (57, 198). The https:// ensures that you are connecting to the It must be considered that for plasmid sequences, evolution can occur not only by mutation and selection but also by horizontal gene transfer. The molecular interactions and the functional relationships that take place in these three types of replication mechanisms are the focus of this review. For example, small plasmids like pRJF1 (2.6 kb) and pWV02 (3.8 kb), isolated from gram-positive bacteria, replicate by the theta mode (115, 152). Priebe S D, Lacks S A. These sequences are used to initiate synthesis of the complementary strand, which converts the ssDNA templates into double-stranded supercoiled circles. For pIP501, it has been shown that steps occurring before stable ctRNA-mRNA duplex formation are sufficient to attenuate mRNA synthesis (34). del Solar G, Daz R, Espinosa M. Replication of the streptococcal plasmid pMV158 and derivatives in cell-free extracts of. This could be done not only through the resolution of crystal structures of protein-DNA complexes but also through the use of many other available physicochemical approaches (such as nuclear magnetic resonance, light and neutron scattering, circular dichroism, and analytical ultracentrifugation, to name a few). Tsutsui H, Fujiyama A, Murotsu T, Matsubara K. Role of nine repeating sequences of the mini-F genome for expression of F-specific incompatibility phenotype and copy number control. For instance, a mutation that affects the LZ-like region of the R6K initiator protein resulted in a protein that failed to activate the or origins of replication (199). Although two IHF-binding sites are flanking the iterons of ori-, the preferential or unique binding site(s) is the one located within the AT-rich region (62, 145a). A 100-bp region in the oriR replication origin is continuously bound by the plasmid RepA protein to form the initiation complex. The DNA sequences of the bind regions are either an inverted repeat contiguous to the nick site (IR-III in the pT181 and pC194 families [Fig. Such a role may not be required for plasmids replicating by the RC mechanism, although the relaxed DNA molecules (which are the products of RC replication) must be supercoiled by DNA gyrase before they become a substrate for a new round of replication. The bind and nic regions of the dso are indicated. 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). 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. Bethesda, MD 20894, Web Policies 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. In: Thomas C M, editor. It should be noted that the presence of directly repeated sequences to which Rep proteins bind is not restricted to plasmids replicating by the theta mechanism, since these sequences have been reported for plasmids using the strand-displacement mechanism or the RC mechanism (171, 176, 267). At an early stage, leading-strand synthesis proceeds in the absence of lagging-strand synthesis (297, 298). Distance matrixes were corrected for multiple substitutions by the method of Kimura. 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. Landschultz W H, Johnson P F, Mcknight S L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Bargonetti J, Wang P Z, Novick R P. Measurement of gene expression by translational coupling: effect of copy mutations on pT181 initiator synthesis. Jovanovic O S, Ayres E K, Figurski D H. Host-inhibitory functions encoded by promiscuous plasmids. Seufert W, Lurz R, Messer W. A novel replicon occurring naturally in. Figure Figure2a2a shows an amino acid alignment of a large family of iteron-binding Rep initiators (encircled in Fig. ori- is in a central position, separated by 3 and 1.2 kb, respectively, from ori- and ori-. Rep proteins encoded by RC-replicating plasmids have several conserved motifs which are shared with the Tra and Mob proteins (involved in plasmid transfer or conjugative mobilization) and with the Rep proteins of ssDNA coliphages and geminiviruses (131, 162, 239, 310). Monomers and dimers of RepA are both functional, but they play different roles: monomers bind to the iterons at the origin, promoting initiation, whereas dimers bind to the adjacent inverted repeat, repressing transcription of the repA gene (181). Bruand C, Ehrlich S D, Jannire L. Unidirectional replication of the structurally stable. Austin S J, Nordstrm K. Partition-mediated incompatibility of bacterial plasmids. The termination of DNA replication is a regulated process, as indicated by the identification of a small protein of E. coli that binds to a terminator site and prevents replication fork arrest mediated by the Tus protein (214). This enhancer partially overlaps ori-, but its activity and the origin function have been distinguished by mutational analysis. The .gov means its official. Plasmids 101: The Promoter Region - Let's Go! - Addgene McEachern M J, Bott M A, Tooker P A, Helinski D R. Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins. The phylogenetic tree groups replicons with similar replicative features: plasmids with Rep proteins binding to iterons (like pPS10, pSC101, R6K, and F) cluster apart from others whose initiators bind to nonrepeated sequences (R1 and its relatives), whereas broad-host-range plasmids (RK2, RA1, {"type":"entrez-protein","attrs":{"text":"RSF10110","term_id":"1534199429"}}RSF10110, and TF-FC2) and replicons with dissimilar initiation mechanisms (phage lambda and phasyl) cluster in separated branches. Thus, the significance of these putative Rep proteins (if any) remains to be clarified. 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. del Solar G, Puyet A, Espinosa M. Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1. Itoh T, Tomizawa J. So, how do you choose? Federal government websites often end in .gov or .mil. Differential inhibition of the DNA translocation and DNA unwinding activities of DNA helicases by the, Hiasa H, Marians K J. Topoisomerase IV can support, Higashi A, Sakai H, Honda Y, Tanaka T, Miao D M, Nakamura T, Taguchi Y, Komano T, Bagdasarian M. Functional features of. A mutation (pcrA3) found in the pcrA gene led to an increase in the proportion of nicked DNA plasmid forms, which were identified as replication initiation complexes (138). DNA synthesis of both strands is coupled and occurs continuously on one of them (leading strand) and discontinuously on the other (lagging strand). The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. RepC protein of plasmid pT181 was purified and shown to have nicking activity on the plasmid dso (156b). The resolution of DNA dimers by specialized systems can also be considered part of the replication termination process (discussed in reference 19). An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. A dimer of seems to bind to each of the seven 22-bp iterons present in ori- (86). Replication of plasmid RSF1010 by the strand displacement mechanism. A quantitative model for control of plasmid mini-F replication in the bacterial cell division cycle. Host replication factors and plasmid initiators form part of the nucleoprotein complex that initiates plasmid replication. The template for initiation of RSF1010 replication is supercoiled plasmid DNA (78, 266). (a) Sequence alignment of the Rep initiator proteins of nine related plasmids of the iteron-containing class. Kes U, Stahl U. Replication of plasmids in gram-negative bacteria. Background. Bldicke T W, Lanka E, Staudenbauer W L. Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE1 plasmid DNA. The study of specific signals involved in the termination of replication of theta-type plasmid replicons is a matter of growing interest. Zavitz K H, Marians K J. Dissecting the functional role of PriA protein-catalyzed primosome assembly in, Zechiedrich E L, Cozzarelli N R. Roles of topoisomerase IV and DNA gyrase in DNA unlinking during replication in. Patel I, Bastia D. A replication origin is turned off by an origin silencer sequence. Iterons are also found in theta-replicating broad-host-range plasmids such as RK2/RP4 (241, 279), pCU1 (164) and pSa (286), as well as in conditional broad-host-range plasmids such as pPS10 (85, 104, 215). Buried in these interrelationships is the ability of plasmids to colonize different hosts (i.e., their host range), which has important ecological and biotechnological implications. A G-type priming signal, located 400 bp downstream of the RepA-binding sequences, has been identified as the site where initiation of the leading strand, primed by DnaG, occurs (186). This suggests that the ssDNAdsDNA conversion requires unpaired sequences within the secondary structures that constitute the sso (65, 68, 109). A mutational analysis has been carried out in the LZ-like motif of the RepA protein of plasmid pPS10 (94). Methylation is not essential for replication, its role being primarily in postreplication (3). Other sites are indicated in the figure. 2B ). I. Bramhill D, Kornberg A. Inhibition by ctRNA is exerted at the posttranscriptional level by hindering the synthesis of the leader peptide. This region also contains the origin of replication, as defined by EM analysis of replication intermediates obtained in vivo (59) and in vitro (266). In some cases, these proteins also participate directly in the generation of the primer. Mller A K, Rojo F, Alonso J C. The level of the pUB110 replication initiator protein is autoregulated, which provides an additional control for plasmid copy number. It has been proposed that the G site is activated when synthesis of the lagging strand, which initiates at the AT-rich region, reaches the G4-like site. ori- contains half an iteron, and ori- one complete iteron that are essential for function. Some of these copy-up mutations were proposed to reduce a strong protein-protein inhibitory interaction induced, at the restrictive temperature, by the thermosensitive mutation (114). The former locus includes the sequences needed for the Rep protein to bind to the plasmid DNA, whereas the latter contains the site where the Rep protein introduces the initial nick (Fig. Koepsel R R, Murray R W, Khan S A. Sequence-specific interaction between the replication initiator protein of plasmid pT181 and its origin of replication. DnaA protein is required for replication from this origin, and it can bind the two dnaA boxes that flank the iterons (146, 147). However, initiation of replication from an internal origin in a plasmid with a terminal protein has been reported (48). This end is elongated by host proteins as the parental strand is being displaced. Nevertheless, implementation of an in vitro system for replication of ssDNA (23, 210) facilitated the determination of the RNA-DNA transition points, which corresponded to regions including the above-mentioned conserved central sequences (CS-6) of the secondary structure of the sso regions (74). GC- and AT-rich regions are also depicted. Due to the genetic organization of the plasmids of the pMV158 family, a similar regulatory circuit seems to exist in all of them (61). Moscoso M, Eritja R, Espinosa M. Initiation of replication of plasmid pMV158: mechanisms of DNA strand transfer reactions mediated by the initiator RepB protein. These three sequences are believed to be melted to allow the DnaBC complex access to the open origin. The intervening sequence between the sites shows potential intrinsic curvature. In addition, these studies support the speculation that the polar arrest of the replication fork, occurring at the Tus-ter complex, could be due to the polar inaccessibility of the helicase to the protein DNA-binding site. Conservation of the nic locus and divergence in the bind region can also be observed in the plasmids of the pMV158 family. Hiraga S I, Sugiyama T, Itoh T. Comparative analysis of the replicon regions of eleven ColE2-related plasmids. The helicase activity of the RepA protein is required during the elongation of RSF1010 replication, and this protein cannot be replaced by the host DnaB helicase. Frey J, Bagdasarian M. The molecular biology of IncQ plasmids. In analogy to ssDNA coliphages, a pRNA is assumed to be needed to initiate lagging-strand synthesis. Directly repeated, 20 bp sequence of plasmid R1162 is required for replication, expression of incompatibility, and copy-number control. During the writing of this review, our labs were financed by Comisin Interministerial de Ciencia y Tecnologa, grants PB94-0127 and PB96-0917 (to R.D.) Honda Y, Sakai H, Komano T, Bagdasarian M. RepB is required, Honda Y, Sakai H, Hiasa H, Tanaka K, Komano T, Bagdasarian M. Functional division and reconstruction of a plasmid replication origin: molecular dissection of the. The pioneering work in Ehrlichs laboratory showed that generation of ssDNA is the hallmark of plasmids replicating by the RC mechanism (108, 291). Model for RC replication. The current model for RepC-catalyzed initiation and termination of pT181 replication (schematized in Fig. RepA mutants having LeuVal substitutions were still able to interact in vitro with the iterons of the origin, indicating that the LZ-like motif is not directly involved in the binding of RepA to DNA. Iterons can also be found outside the origin region in some plasmids (P1, F, RK2, R6K, Rts1, and pColIV-K30). Wang P-Z, Projan S J, Henriquez V, Novick R P. Origin recognition specificity in pT181 plasmid is determined by a functionally asymmetric palindromic DNA element. Kozlowski M, Thatte V, Lau P C K, Visentin L P, Iyer V N. Isolation and structure of the replicon of the promiscuous plasmid pCU1. Control of replication by inhibitors would require that they could measure the concentration of plasmid copies within the cell. Giraldo R, Martn M, Fernndez-Tresguerres M E, Nieto C, Daz R. Mutations within the minimal replicon of plasmid pPS10 increase its host range. Brantl S, Behnke D, Alonso J C. Molecular analysis of the replication region of the conjugative. oriR, defined as the minimal region required for RepA-dependent replication of R1 in vitro, is bound specifically by RepA (101, 183, 184). This contrasts with the situation for plasmids replicating by the strand displacement mechanism, in which the same plasmid-encoded primase and helicase proteins are involved in replication of both strands, in a continuous process that starts in two symmetrical origins and proceeds in opposite directions (263, 266). Replication occurs with opposite polarities from two origins (ssiA and ssiB), which are independently used. Nez B, Avila P, de la Cruz F. Genes involved in conjugative DNA processing of plasmid R6K. Origin of Replication - an overview | ScienceDirect Topics As the result of an odd number of homologous recombination events, dimers can arise. This is accomplished by various proteins (initiators and repressors) binding to the sequence that make up the ori. Levchenco I, Filutowicz M. Initiator protein Pi can bind independently to two domains of the gamma origin core of plasmid R6K: the direct repeats and the A+T-rich segment. In vivo and in vitro replication of R1 requires the initiator protein, RepA (77, 159, 183, 305). H4S47 O-GlcNAcylation regulates the activation of mammalian replication Giraldo-Surez R, Fernndez-Tresguerres M E, Daz-Orejas R, Malki A, Kohiyama M. The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication. With some exceptions, plasmids using the theta mechanism of replication require a plasmid-encoded Rep initiator protein. (iii) The third is the plasmid-borne genes involved in the control of replication. Origin of replication pUC (500 copies) Peptide cleavage no cleavage Promoter Promoter name: CMV Promoter activity: constitutive Promoter type: mammalian reporter gene none shipped in ambient storage temp. Zillig W, Kletzin A, Schleper C, Holz I, Janekovick D, Hain J, Lanzendrfer M, Kristjansson J K. Screening for Sulfolobales, their plasmids and their viruses in Icelandic solfataras. They can incorporate and deliver genes by recombination or transposition, thus favoring genetic exchanges in bacterial populations. Theta-type DNA synthesis can start from one or from several origins, and replication can be either uni- or bidirectional. In vitro and in vivo indications that the atypical ribosome-binding site is involved in the translation of the pMV158 repB gene has been obtained (70). There is a set of two tandem dnaA boxes at one end and a set of three tandem dnaA boxes at the other end of the P1 origin. 3. 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. Grohmann E, Zechner E L, Espinosa M. Determination of specific DNA strand discontinuities with nucleotide resolution in exponentionally growing bacteria harbouring rolling circle-replicating plasmids. The amount of aeration, temperature, culture volume, antibiotic, and medium can all affect copy number. 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). They consist of a stretch of 47 bp (ColE2) or 33 bp (ColE3) and contain two major directly repeated sequences. Rep-mediated nicking participates in the initiation of replication because the Rep proteins cleave supercoiled DNA within an unpaired sequence of the nic region. Hybrid formation between the antisense and the preprimer RNA alters the overall secondary structure of the preprimer. A typical feature of the RC-replicating plasmids is that the nic regions are highly conserved among replicons of the same family whereas differences are found at the bind loci. . - Studybuff An origin of replication is a sequence of DNA at which replication is initiated on a chromosome, plasmid or virus. A hypothetical role for DnaK in modulating the aggregation and activation state of RepA dimers, inspired by the findings for P1 plasmid RepA initiator (316), is also shown. The ori is the place where DNA replication begins, enabling a plasmid to reproduce itself as it must to survive within cells. Bernander R, DasGupta S, Nordstrm K. The. The control circuit elements (copG and/or rnaII) have been cloned at a physiological gene dosage (i.e., at a copy number similar to that of the wild-type plasmid). Moscoso M, del Solar G, Espinosa M. Specific nicking-closing activity of the initiator of replication protein RepB of plasmid pMV158 on supercoiled or single-stranded DNA. Bastia D, Mohanthy B K. Mechanisms for completing DNA replication. Manen D, Upegui-Gonzalez L C, Caro L. Monomers and dimers of the RepA protein in plasmid pSC101 replication: domains in RepA. The EMBL database accession numbers are as follows: pPS10, {"type":"entrez-nucleotide","attrs":{"text":"X58896","term_id":"45872"}}X58896; pECB2, {"type":"entrez-nucleotide","attrs":{"text":"Y10829","term_id":"1808661"}}Y10829; pRO1614, {"type":"entrez-nucleotide","attrs":{"text":"L30112","term_id":"463167"}}L30112; pCM1, {"type":"entrez-nucleotide","attrs":{"text":"X86092","term_id":"769802"}}X86092; pFA3, {"type":"entrez-nucleotide","attrs":{"text":"M31727","term_id":"148533"}}M31727; pSC101, {"type":"entrez-nucleotide","attrs":{"text":"K00828","term_id":"150939"}}K00828; pCU1, {"type":"entrez-nucleotide","attrs":{"text":"Z11775","term_id":"43899"}}Z11775; RepFIA, {"type":"entrez-nucleotide","attrs":{"text":"Y00547","term_id":"45794"}}Y00547; R6K, {"type":"entrez-nucleotide","attrs":{"text":"M65025","term_id":"151841"}}M65025. Pak M, Wickner S. Mechanism of protein remodeling by ClpA chaperone. Origins of replication and copy numbers of various plasmids and cosmids * The pMB1 origin of replication is closely related to that of ColE1 and falls in the same incompatibility group. Once the checkpoint corresponding to the initiation of leading-strand synthesis is past, replication continues until completion, following a process catalyzed by DNA Pol III and other host proteins. Lin J, Helinski D R. Analysis of mutations in. Mutations in copA that decrease the efficiency of the complex formation increase the copy number, but replication still is controlled. To generate the replication initiation complex of pT181, the product of the Staphylococcus aureus chromosomal pcrA gene is required. This is achieved by plasmid-encoded control elements that regulate the initiation of the replication step. Cereghino J L, Helinski D R. Essentiality of the three carboxyl-terminal amino acids of the plasmid RK2 replication initiation protein TrfA for DNA binding and replication activity in gram-negative bacteria. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. The latter proteins could contribute to the dissociation of the RepA dimers into monomers, which is the form of the RepA protein that recognizes the five iterons of the origin (58, 315). In vitro analyses have shown that IR-II of plasmid pC221 (closely related to pT181) or hairpin I of pMV158 are sufficient for the nicking-closing activity of their respective Rep proteins. An auxiliary role is provided by a small basic protein, termed Rop or Rom (276, 304). Although most of the RC-replicating plasmids so far described are smaller than 10 kb, all small plasmids do not necessarily replicate by the RC mode. Kiewiet R, Bron S, de Jonge K, Venema G, Seegers J F M L. Theta replication of the lactococcal plasmid pWVO2. 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]). There are lots oforigins of replicationout there so, for simplicitys sake, we've ignored those used in eukaryotic cells and viruses and focused only on those found in bacteria. The origin of replication of plasmid RSF1010 has been defined as the minimal region able to support bidirectional replication when the RSF1010 replication proteins (RepA, RepB, and RepC) are supplied in trans by a second plasmid (266). Interactions of RepA protein with the oriR region promote, both in vitro and in vivo, initiation of leading-strand synthesis at a DnaG-priming site (the G site, resembling the bacteriophage G4 origin for complementary strand synthesis) (21, 186) that is located 400 bp downstream of oriR. subculture directly from glycerol stocks, agar stabs, or liquid cultures. Similar situations were also found for other iteron-containing plasmids, such as P1 or R1162/RSF1010. 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).
