Gel filtration studies have shown that the apparent molecular weight of RepC is consistent with dimer formation (257). DnaA is required for the delivery of the DnaB helicase to the origin region and both DnaA and TrfA are required for DnaB-induced template unwinding. It is always good to k, eep in mind that plasmids with low to medium copy numbers can still express massive amounts of protein given the proper, As a rule, plasmids from the same group should not be co-transformed. The replicon is comprised of the origin of replication (ori) and all of its control elements. The current model for RepC-catalyzed initiation and termination of pT181 replication (schematized in Fig. Mechanistically, the situations for pUB110 and the pMV158 may be indistinguishable, which may point to a more general control of replication mechanism in these plasmids replicating by the RC mode. In: Molineux I, Kohiyama M, editors. 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. In this review, we focus on replication and its control in circular plasmids. Members of this family require three plasmid-encoded proteins for initiation of DNA replication. 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. 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. Kamio Y, Terawaki Y. Nucleotide sequence of an incompatibility region of mini-Rts1 that contains five direct repeats. Bird R E, Tomizawa J. Ribonucleotide-deoxyribonucleotide linkages at the origin of DNA replication of colicin E1 plasmid. Plasmid segregation is maintained by a par locus-a partition locus that ensures each daughter cells gets on plasmid. In addition, ctRNA prevents the formation of an activator RNA pseudoknot that is required for efficient synthesis of the Rep protein (reviewed in reference 309). Brantl S, Birch-Hirschfeld E, Behnke D. Rep protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism. The transcript folds into a secondary structure which stabilises the interaction between the nascent RNA and the origin's DNA. Pansegrau W, Lanka E. Common sequence motifs in DNA relaxases and nick regions from a variety of DNA transfer systems. Most of the chromosomes of prokaryotic cells or bacteria, including E. Coli, have a supercoiling circular form. Deletion of plasmid sso regions leads to reduction in the plasmid copy number (measured as dsDNA), accumulation of ssDNA molecules, and rapid loss of the plasmid in the absence of selective pressure (65, 109). One of the most relevant features of RC replication is that the newly synthesized leading plus strand remains covalently bound to the same parental plus strand. This regulatory system is independent of the one modulated by iterons. I. Bramhill D, Kornberg A. A two-component system (Cop protein and antisense RNA) controlling the replication of plasmid pIP501 has also been proposed (35; see below). Methylation is not essential for replication, its role being primarily in postreplication (3). Vocke C, Bastia D. DNA-protein interaction at the origin of DNA replication of the plasmid pSC101. A similar model has been postulated for other plasmid Rep proteins, in which monomeric and dimeric forms of the protein are involved in replication and autoregulation, respectively (discussed in reference 51). The origin of vegetative replication is one of the most important elements in plasmid. Ilyina T V, Koonin E V. Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes, and archaebacteria. 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). When autoregulation exists, either a single species of the protein is involved in both regulation and replication, or different species of the protein, monomeric and dimeric, recognize the origin and the regulatory regions, respectively (discussed in reference 51). An origin of replication is a sequence of DNA at which replication is initiated on a chromosome, plasmid or virus. 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). 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). The control of plasmid replication is plasmid encoded and is performed by molecules (antisense RNA, proteins, or DNA sequences) that inhibit the initiation of replication in a dose-dependent way. Opening of the strands is catalyzed by specific initiators (Rep and DnaA proteins) and/or by transcription by RNAP. However, no alternative model for leading-strand termination has been proposed, and inactivation of the RepB protein has not been analyzed. 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]). Fig.3).3). Bernander R, DasGupta S, Nordstrm K. The. DNA replication: the regulatory mechanisms. Conversion of ssDNAdsDNA from an intact ssoW seems to require the host RNAP (173). Adams D E, Bliska J B, Cozzarelli N R. Cre-, Alonso J C, Leonhardt H, Stiege C. Functional analysis of the leading strand replication origin of plasmid pUB110 in, Alonso J C, Taylor R H. Initiation of plasmid pC194 replication and its control in. The end products of this reaction would be a dsDNA plasmid molecule (containing the newly synthesized plus strand) and a RepC/C* heterodimer composed of one intact RepC molecule and one modified subunit which has a short oligonucleotide covalently bound. The iteron-containing origin (oriV) and motifs found in the replication initiator protein (RepA) are depicted. Plasmid origins of replication can be defined as (i) the minimal cis-acting region that can support autonomous replication of the plasmid; (ii) the region where DNA strands are melted to initiate the replication process, or (iii) the base(s) at which leading-strand synthesis starts. The orientation of the dnaA boxes and the different sensitivity of the two strands to reagents specific for single-stranded DNA suggest that DnaA-dependent loading of DnaB preferentially occurs in one of the strands, which can account for the unidirectional mode of P1 replication (206). The second element regulating pIP501 copy number is the unusually long-lived antisense RNA III (35). Fig.1.1. Helicase unwinds the plasmid DNA and relaxase attaches to the transfer DNA strand. This leads to a transient increase in the transcription rate of repA and, as a consequence, to a temporal increase in the frequency of plasmid replication. McEachern M J, Filutowicz M, Yang S, Greener A, Mukhopadyay P, Helinski D R. Elements involved in the copy number regulation of the antibiotic resistance plasmid R6K. The two characteristic motifs found in the Rep initiators, LZ (hydrophobic heptad residues pointed to by open arrowheads) and HTH, are indicated. A mutational analysis has been carried out in the LZ-like motif of the RepA protein of plasmid pPS10 (94). Protein binds efficiently to the iterons of the ori- but not to ori- or ori-. Plasmids with phage-derived ori's are referred to as phagemids. As a library, NLM provides access to scientific literature. Some replicons may require the host DNA polymerase I (DNA Pol I) during the early stages of leading-strand synthesis. Two distinct mechanisms of synthesis of DNA fragments on colicin E1 plasmid DNA. and grant BIO97-0347 (to M.E.). Churchward G, Linder P, Caro L. The nucleotide sequence of replication and maintenance functions encoded by plasmid pSC101. Yasukawa H, Hase T, Sakai A, Masamune Y. Rolling-circle replication of the plasmid pKYM isolated from a Gram-negative bacterium. Several palindromes flanking a putative dnaA box are located within the origin of replication of pLS20. 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). 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). This determines a constant rate of replication, independent of the copy number of the plasmid or the growth rate of the host. Conclusions: RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. Plasmid R1 is the most extensively studied member of the IncFII family of plasmids. Gros M-F, te Riele H, Ehrlich S D. Rolling circle replication of single-stranded DNA plasmid pC194. Three highly conserved sequence patches are found within the iterons of this replicon. This indicates that the mutation has affected a protein-protein interface and suggests that this interface could be located in the C-terminal region of RepA. RC replication was originally thought to be limited to ssDNA coliphages and to small multicopy plasmids isolated from gram-positive bacteria. Haring V, Scholz P, Scherzinger E, Frey J, Derbyshire K, Hatfull G, Willetts N S, Bagdasarian M. Protein RepC is involved in copy number control of the broad host range plasmid RSF1010. Revised versions of the replicon model for all domains of life. An 18-base pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181. Natarajan S, Kaul S, Miron A, Bastia D. A 27 kd protein of, Nieto C, Giraldo R, Fernndez-Tresguerres E, Daz R. Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for. 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). Priming of DNA synthesis at these origins is catalyzed by the plasmid-specific primase (RepB). 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). During the final stages of plasmid replication, catenates containing gaps in both daughter strands can be originated (212). These replicons have been found in species from the three representatives of the living world, namely, the domains Archaea, Bacteria, and Eukarya (318). Compilation and comparative analysis. Plasmids 101, Plasmid has been documented in both circular and linear form but most known and common plasmids are circular. 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. Seufert W, Dobrinski B, Lurz R, Messer W. Functionality of DnaA protein binding site in DNA replication is orientation-dependent. Overproduction of the pUB110-Rep protein showed only one major product, the one translated from the first Met residue (178). Antisense RNA has been involved in the silencing of the ori- of plasmid R6K (243, 244). Alignment of iterons present in the origin of replication of different plasmids showed the conservation of the hexanucleotide TCAGPuG (86), which is directly involved in the binding of the initiator protein to the ori- region of plasmid R6K (97, 98). Although the sequence and regulation of theoridramatically affect the copy number of a plasmid, other external factors contribute as well. 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). ColE1 replication begins at the origin. Some plasmids carry and oriT: origin of transfer. The structural definition of these macromolecular assemblies will provide mechanistic information relevant to our understanding of the initiation of plasmid replication and the interrelations between plasmids and their hosts. rep mutants leading to impaired Rep protein-DNA interactions have been found in various plasmids. An official website of the United States government. With some exceptions, plasmids using the theta mechanism of replication require a plasmid-encoded Rep initiator protein. A second solution to the titration/autoregulation paradox was provided by the model that Rep molecules bound to the P1 iterons were still able to repress the repA promoter (49). This last reaction will release the ssDNA intermediate. Other symbols as follows: solid ellipses, origins; rectangles, promoters; a.r.b.s., atypical ribosome-binding site; parallel vertical lines, mRNA-ctRNA interactions; plus, positive interactions; minus, and inhibitory RNA-RNA or protein-DNA interactions. Hybrid formation between the antisense and the preprimer RNA alters the overall secondary structure of the preprimer. The convergence of structural studies with the functional analysis of initiators and inhibitors of DNA replication is an important area of future development. Brendler T, Abeles A, Austin S J. The requirement of a plasmid-encoded initiator is reflected by the presence of DNA cognate sites in the origin of replication, where protein-DNA interactions take place. Holmes M L, Pfeifer F, Dyall-Smith M L. Analysis of the halobacterial plasmid pHK2 minimal replicon. This can be achieved by Rep-facilitated extrusion of a cruciform structure encompassing the nic region of the origin, where the nick site is unpaired. This motif has been described in many prokaryotic DNA-binding proteins, where it is involved in binding to specific regulatory DNA regions (39, 235). Over the sequences is shown a secondary-structure prediction performed by the neural network algorithm PHD (260) on the output from CLUSTAL W: predicted -helical regions (boxes) and -strands (arrows). Dellis S, Filutowicz M. Integration host factor of. Among the two mechanisms, replication can occur by any one of the mechanisms: Koonin E V, Ilyina T V. Computer-assisted dissection of rolling circle DNA replication. The function of the middle patch is less clear, but it may contribute to a proper conformation of the RepA-binding site. Individual plasmid copies are selected for replication at random from a pool that includes replicated and nonreplicated copies. 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). Important information on the initial events of replication of plasmid RK2 has been obtained (161a). A region of the pPS10-RepA protein probably involved in protein-protein interactions with host replication factors has been identified. Three different types of nic regions, belonging to (i) the pT181 family, similar to that of M13, (ii) the pC194 family, exhibiting similarities to X174, and (iii) the pMV158 family, with no relevant homologies to nic regions from known ssDNA coliphages, have been reported (Table (Table1)1) (69, 108, 228). The sequestration is relieved as the hemimethylated DNA undergoes new methylation (51). Replication and Control of Circular Bacterial Plasmids - PMC Dempsey L A, Birch P, Khan S A. Tanaka K, Kino K, Taguchi Y, Miao D M, 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. Dam methylation sequences are not present in other plasmid replicons. Theta-type replication is, in most cases, unidirectional. Larger DNAs have many origins, and DNA An origin of replication is a sequence of DNA at which replication is initiated on a chromosome, plasmid or virus. It is packaged in its viral capsid as a single stranded DNA genome. The RepA helicase of RSF1010 works in the 53 direction, which implies that it is working while bound to the displaced strand. Unlike Tus, which acts as a monomer, RTP acts as a tetramer of two dimers (261, 262). Furthermore, once a RepC dimer has been used, it becomes inactive for a new round of replication because of the attachment of an oligonucleotide to one of its subunits, generating a heterodimer, RepC/C* (255257). 555bp upstream from this point, RNA polymerase initiates transcription of RNAII which acts as a pre-primer and begins the synthesis of the leader strand. Plasmid pUB110 and its relative pRBH1 were reported to contain two putative SD sequences followed by a Met residue (178, 208). The ter sequence is the binding site of Tus, a monomeric protein of E. coli that promotes the termination of plasmid replication (121, 275). The replication intermediates can also be monitored by one- or two-dimensional electrophoresis. Seegers J F M L, Zhao A C, Meijer W J J, Khan S A, Venema G, Bron S. Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01. Abeles A L, Reaves L D, Youngre-Grimes B, Austin S J. The organization of this sequence as two separable and polar terminus sites was recognized and verified (130). Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Jannire L, Bruand C, Ehrlich S D. Structurally stable DNA cloning vectors. Sites 1 and 2 share a core sequence (g/tTTAAA) that is an imperfect palindrome (101). Wojtkowiak D, Georgopoulos C, Zylicz M. Isolation and characterization of ClpX, a new ATP-dependent specificity component of the Clp protease of. Scherzinger E, Haring V, Lurz R, Otto S. Plasmid RSF1010 DNA replication. Plasmid origins of replication can be defined as (i) the minimal cis-acting region that can support autonomous replication of the plasmid; (ii) the region where DNA strands are melted to initiate the replication process, or (iii) the base(s) at which leading-strand synthesis starts. These considerations are especially useful to keep in mind if you are planning to purify your plasmid DNA: Have any questions? Novick R P, Adler G K, Projan S J, Carleton S M, Highlander S K, Gruss S A, Khan S A, Iordanescu S. Control of pT181 replication. The assembly of the preinitiation complex and details of the molecular interactions leading to the initiation of replication are well documented for a few theta-replicating plasmids (described below). The origin was included in many plasmids to enable isolation of. 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. For cells of each domain type, trans-acting initiators recognize replication origins to assemble prereplicative complexes required to unwind the DNA and load DNA helicase.Eukaryotic initiators are preassembled into hexameric origin recognition complexes (ORCs) before interacting with DNA. The proximal half of IR-III is more important for RepC recognition of the dso than is the distal half. 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. Khan S A. Note the A -C compatibility grouping is an arbitrary designation, and plasmids from the same incompatibility group should not be co-transformed. F1 is a phage-derived ori that allows for the replication and packaging of ssDNA into phage particles. The pPS10 replicon also contains the repA gene, encoding the RepA initiator protein (shadowed ovals). The nucleotide sequence of the ter region was determined, and the replication terminus of R6K was cloned (17, 18). This is accomplished by various proteins (initiators and repressors) binding to the sequence that make up the ori. Mechanism of replication and copy number control of plasmids in Gram-positive bacteria. Identification of a common fold in the replication terminator protein suggests a possible model for DNA binding. This could represent a mechanism for regulation of the level of active replication protein (87). The new mRNA configuration would be such that a new hairpin ending in an A(U)6 sequence could form. ColE1 is the prototype of a class of small multicopy plasmids that replicate by a theta-type mechanism. Replication starts when these origins are exposed as single-stranded regions. 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. pUC18 is one of the most widely used cloning and expression vectors within research labs today. These plasmids will also carry functions needed to be mobilized or mob genes. Sequences that were virtually identical (pairwise scores, 90) were discarded, and a refined alignment was used as input data for the DISTANCES program of the Genetics Computer Group software package (95). These analyses provide information on the nature of the replication intermediates, direction of replication, location of the origin and terminus, and degree of coupling between leading- and lagging-strand synthesis. Specific recognition between the Rep protein and the dso is required not only for initiation of RC replication but also for the termination step. Plasmid - an overview | ScienceDirect Topics If a plasmid's replication rate is too slow it will eventually be lost; however, a high rate of replication is also undesirable as it is a burden . Kittell B L, Helinski D R. Plasmid incompatibility and replication control. Multiple iterons are required for origin activity, although not all iterons present in a given origin have to be essential. These interactions are needed for replication, as indicated by the defective replication phenotype associated with a repA mutant that failed to generate high-order RepA-oriR complexes (101, 232). Twigg A J, Sherrat D. Trans-complementable copy-number mutants of plasmid ColE1.
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