Filutowicz M, MacEachern M J, Helinski D R. Positive and negative roles of an initiator protein at an origin of replication. Moscoso M, Eritja R, Espinosa M. Initiation of replication of plasmid pMV158: mechanisms of DNA strand transfer reactions mediated by the initiator RepB protein. Some steps in these directions have recently been taken for the RepA protein of plasmid P1 (50). The bind and nic regions of the dso are indicated. In addition, plasmids may bear genes that could be considered dispensable, although they could actually play an important role for the plasmid itself and/or for the host. Holmes M L, Pfeifer F, Dyall-Smith M L. Analysis of the halobacterial plasmid pHK2 minimal replicon. Inhibition by ctRNA is exerted at the posttranscriptional level by hindering the synthesis of the leader peptide. This elegant approach led pC194-based plasmids to reinitiate after one round of replication, in a similar fashion to the replication of X174 DNA (218). Where does pUC18 come from? View history Models for bacterial ( A) and eukaryotic ( B) DNA replication initiation. Lacks S A, Lpez P, Greenberg B, Espinosa M. Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. RepA protein of R1 seems to interact initially with the two partially palindromic sequences at the oriR region as a dimer, but further binding to the intermediate region could be by monomers, since there are no symmetrical sequences (102). 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). Koepsel R R, Khan S A. Static and initiator protein-enhanced bending of DNA at a replication origin. Specific recognition between the Rep protein and the dso is required not only for initiation of RC replication but also for the termination step. A single-strand initiation site (ssi) for the priming of DNA replication has been located near the ori of ColE2 (219). Khatri G S, MacAllister T, Sista P R, Bastia D. The replication terminator protein of. 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. . Once the primosome is assembled at the pas site, it translocates in the 53 direction, unwinding the helix and priming the discontinuous DNA synthesis. The ssoW of plasmid pWV01 is made up of two inverted repeats (IR1 and IR2). Conley D L, Cohen S N. Effects of the pSC101 partition (, Conley D L, Cohen S N. Isolation and characterization of plasmid mutations that enable partitioning of pSC101 replicons lacking the partition (. Replication of the pAM1/pIP501 broad-host-range plasmid family from gram-positive bacteria (32, 41, 42) requires, like ColE2 and ColE3, DNA Pol I and Rep protein. In plasmid pSC101, the last third of the RepA protein is not needed for binding to specific DNA sites (181), which contrasts with the role of the C-terminal region in other initiators. Whether those features of the translation initiation signals of the rep genes reflect an additional control mechanism to keep a low level of the Rep protein is not known. The complete nucleotide sequence of a small cryptic plasmid from a rumen bacterium of the genus, Hiasa H, Marians K J. Three independently isolated mutations were located within the region encoding the LZ motif of RepA. 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). It has been proposed that in RK2 these auxiliary iterons stimulate initial pairing between origins. 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). This plasmid-host communication has attracted the attention of researchers working in environmental and in evolutionary fields. Mutational analyses have shown that the nucleotides of the right arm of the inverted repeat IR-II must be conserved for termination to occur whereas changes are allowed in some of the nucleotides of the left arm of the IR-II (330). Wickner S, Hoskins J, McKenney K. Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA. Independence of tight substrate binding and catalytic activity of relaxase (TraI) of IncPa plasmid RP4. Bldicke T W, Lanka E, Staudenbauer W L. Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE1 plasmid DNA. Devine K M, Hogan S T, Higgins D G, McConnell D J. Replication and segregational stability of the. These sequences are used to initiate synthesis of the complementary strand, which converts the ssDNA templates into double-stranded supercoiled circles. 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.) Iterons or dnaA boxes typical of the theta-type replicons have not been found in phasyl (271). Eckdahl T T, Anderson J N. Conserved DNA structures in origins of replication. The origins of replication ( ori) are plasmid-borne DNA sequences that direct the host cell to initiate plasmid replication and are thus essential for plasmid propagation. In many cases, the origin of replication contains directly repeated sequences, termed iterons, which are the binding sites for the plasmid-encoded Rep proteins and which have control properties. RepA monomers contact each iteron through two consecutive major grooves on the same face of the DNA helix (242). Region of the streptococcal plasmid pMV158 required for conjugative mobilization. (b) Phylogenetic tree for theta-type replicons from gram-negative bacteria, based on sequence alignments of their Rep proteins (such as the one shown in panel a for the pPS10 family, encircled in the tree with a dashed line). Abeles A L, Reaves L D, Youngre-Grimes B, Austin S J. Intrinsic DNA bends at the Rep-binding sites would favor additional curvatures of the origin induced by Rep proteins. Two early events in this mode of replication are the opening of the strands at specific sequences (the origin of replication) and the synthesis of RNA primers. oriR, defined as the minimal region required for RepA-dependent replication of R1 in vitro, is bound specifically by RepA (101, 183, 184). Similar situations were also found for other iteron-containing plasmids, such as P1 or R1162/RSF1010. Hill T M. Arrest of bacterial DNA replication. The two characteristic motifs found in the Rep initiators, LZ (hydrophobic heptad residues pointed to by open arrowheads) and HTH, are indicated. The plasmid replication origin is often named oriV for ori vegetative, to distinguish it from oriT. Fig.3).3). 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. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semiconservative replication prior to cell division to ensure each daughter cell receives the full complement of chromosomes. 20C Description General description A versatile cloning vector for the expression of genes in mammalian cells. 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). In: Helinski D R, Cohen S N, Clewell D B, Jackson D A, Hollaender A, editors. as a modification of an existing group of cloning plasmids. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This table defines common cloning vectors, their copy number, ori, and incompatibility groups. II. Bidirectional DNA replication, in which two replication forks travel in opposite directions from each origin of replication, is required for complete genome duplication. RepA variants that fail to repress the repA promoter had amino acid changes within or in the vicinity of an HTH motif located at the C-terminal end of the protein (93). Blomberg P, Wagner E G H, Nordstrm K. Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. Kleanthous H, Clayton C L, Tabaqchali S. Characterization of a plasmid from. copA is transcribed from a constitutive promoter, and its product is an unstable (half-life, 2 min) RNA, CopA, which is complementary to a leader region (termed CopT) of the repA mRNA. 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). Brendler T, Abeles A, Austin S J. Also, let us know if you have any special requests for future Plasmids 101 series topics and stay tuned for the next installment. Sakai H, Komano T. DNA replication of the IncQ broad-host-range plasmids in gram-negative bacteria. Acebo P, Alda M T, Espinosa M, del Solar G. Isolation and characterization of pLS1 plasmid mutants with increased copy numbers. The origin of vegetative replication is one of the most important elements in plasmid. 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. Protein conformational changes are coupled to the dissociation of RepA dimers (which have a compact package of both domains) into monomers (with an elongated arrangement of the domains). 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. Blomberg P, Nordstrm K, Wagner E G H. Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation. The phylogenetic tree was built up according to the UPGMA method with the program GROWTREE (95). 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. 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. Maps are based on the following references: pPS10 (104, 215); pSC101 (132, 284); P1 (6); RK2 (278); R6K (277); R1 (101); ColE1 (280, 301); ColE2-type plasmids (124). This is known as incompatibility. Iterons can be adjacent or separated by intervening sequences. 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. 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). Kim K, Meyer R J. Bolivar F, Rodriguez R I, Betlach M C, Boyer H W. Ampicillin-resistant derivatives of the plasmid pMB9: construction and characterization of new cloning vehicles. The sequences for 35 initiators were retrieved from databases, and a preliminary alignment was performed with CLUSTAL W (data not shown). Under electron microscopy (EM), the replication intermediates are seen as typical (theta)-shaped molecules that, when digested with enzymes that cleave within the replicated region, yield Y-shaped molecules (forks). True (Restriction digestion produces fragments of DNA and the sizes of these fragments can be determined by gel electrophoresis using standard DNA fragments of know size.) Puyet A, del Solar G, Espinosa M. Identification of the origin and direction of replication of the broad-host-range plasmid pLS1. Cross M A, Warne S R, Thomas C M. Analysis of the vegetative replication origin of broad-host-range plasmid RK2 by transposon mutagenesis. RepA protein, probably as a dimer, recognizes sequentially (albeit with different affinities) the cores of two partially palindromic sequences (Fig. Requirement for topoisomerase I in the maintenance of template specificity. Replication intermediates can provide ideal substrates for these homologous recombination events. Noirot-Gros M F, Bidnenko V, Ehrlich S D. Active site of the replication protein of the rolling circle plasmid pC194. Yasueda H, Takechi S, Sugiyama T, Itoh T. Control of ColE2 plasmid replication: negative regulation of the expression of the plasmid-specified initiator protein, Rep, at a posttranscriptional step. Origins of replication without iterons can be found in other well studied theta-replicating plasmids like R1 and ColE1, as well as in plasmid pLS20 from B. subtilis. The site is secure. The intervening sequence between the sites shows potential intrinsic curvature. Ingmer H, Cohen S N. Excess intracellular concentration of pSC101 RepA protein interferes with both plasmid DNA replication and partitioning. Control of P1 plasmid replication by iterons. The RepA protein is a hexamer of 30-kDa subunits, and it contains two activities: an ssDNA-dependent ATPase and a 53 DNA helicase. The biotechnological and clinical implication of this important discovery is well known and reveals, once more, the important practical implication of basic studies. This suggests that the ssDNAdsDNA conversion requires unpaired sequences within the secondary structures that constitute the sso (65, 68, 109). During the final stages of plasmid replication, catenates containing gaps in both daughter strands can be originated (212). Peng H, Marians K J. Decatenation activity of topoisomerase IV during. Dibbens J A, Muraiso K, Chattoraj D K. Chaperone-mediated reduction of RepA dimerization is associated with RepA conformational change. Flashner Y, Shlomai J, Shafferman A. The melting of the DNA strand is dependent on two plasmid replication proteins, RepC and RepA, and is facilitated by an AT-rich region that precedes the ssiA and ssiB regions. The RepB protein of pMV158 binds in vitro to a dsDNA fragment containing the direct repeats (60). de la Campa A G, del Solar G, Espinosa M. Initiation of replication of plasmid pLS1. The question of the role of autoregulation remains open, and no simple answer has been provided. The classic way of classifying plasmids is to distribute them among incompatibility groups, whose members have very similar origin sequences and replication control mechanisms. Pouwels P H, van Luijk N, Leer R J, Posno M. Control of replication of the. Is the gene toxic in high amounts? Once you've gotten to know what a plasmid is in general, it's time to take a look at some of its parts. Furthermore, two other residues (Glu-142 and Glu-210) are also required for the catalytic activity (217). 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. In vitro studies have shown that an ssDNA fragment containing the pMV158-ssoA was able to promote RNAP-directed synthesis of a 20-nucleotide pRNA. Control of mini-R1 plasmid replication: a computer simulation. In spite of the progress that has been made in the study of plasmid replication and its control, there are major gaps in our knowledge of the various replication systems. The replication initiator protein, , seems to favor interactions between these transcripts. Fig.2a2a support a similar structural organization for other Rep proteins of theta-replicating plasmids. None of the Rep proteins seems to have the HTH motif typical of many DNA-binding proteins, although a putative LZ motif exists in the RepB protein of plasmid pMV158 (60). Tomizawa J. Replication of colicin E1 plasmid DNA in vitro. Inspection of the DNA sequence of various rep genes shows two possible translation initiation signals, which would give rise to Rep and Rep proteins (similar to the A and A* initiator proteins, respectively, of the ssDNA coliphage X174) (13). 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. Germino J, Bastia D. Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA. Lin L S, Meyer R J. These events determine the unidirectional pattern of ColE1 replication. 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). In addition, DNA Pol I can participate in the early synthesis of the leading strand (ColE1 and pAM1). 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).
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