INTRODUCTION

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Plasmids are extrachromosomal DNA elements with characteristic copy numbers within the host. These replicons have been found in species from the three representatives of the living world, namely, the domains Archaea, Bacteria, and Eukarya (318). Plasmids may constitute a substantial amount of the total genetic content of an organism, representing more than 25% of the genetic material of the cell in some members of the Archaea (127, 331). They can incorporate and deliver genes by recombination or transposition, thus favoring genetic exchanges in bacterial populations. Since plasmids can be introduced into new hosts by a variety of mechanisms, they can be considered to be a pool of extrachromosomal DNA which is shared among populations. The wealth of genetic information carried by plasmids, their impact in the microbial communities, and the potential of these elements to act as natural cloning vectors have stimulated research into plasmids not only from the fundamental but also from the clinical, biotechnological, and environmental points of view. Three main factors have contributed to the development of plasmid research: (i) the genetic organization of these elements is apparently simple, (ii) they can be easily isolated and manipulated in vitro, and (iii) since plasmids are dispensable, their manipulation does not appear, in principle, to have adverse consequences to the hosts.

The feature that better defines plasmids is that they replicate in an autonomous and self-controlled way. The analysis of plasmid replication and its control has led to milestone discoveries, such as the existence of antisense RNAs, and has contributed to the unraveling of mechanisms of DNA replication, macromolecular interactions, and control of gene expression. 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. This plasmid-host communication has attracted the attention of researchers working in environmental and in evolutionary fields. Plasmid host range studies also have clear implications in clinical microbiology and in biotechnology. Despite their autonomous replication, plasmids extensively use the replication machinery of the host, and therefore plasmid replication studies facilitate the exploration of the mechanisms involved in chromosome replication.

There are three general replication mechanisms for circular plasmids, namely, theta type, strand displacement, and rolling circle (RC). Historical development of research on plasmids has led to the idea that theta replication is more frequent in replicons from gram-negative than from gram-positive bacteria whereas the opposite is found for plasmids replicating by the RC mode. This belief is probably wrong. It is true, however, that present knowledge on theta-replicating plasmids stems from replicons from gram-negative bacteria and that on RC-replicating plasmids derives from replicons from gram-positive hosts. Strand displacement replication has been associated with broad-host-range plasmids from the IncQ family. The molecular interactions and the functional relationships that take place in these three types of replication mechanisms are the focus of this review. Linear plasmids have been found in both gram-positive and gram-negative bacteria, and their structure can be of two types: those having a hairpin at each end, and those having a protein covalently bound at their 5' ends. Linear plasmids of the first group replicate via concatemeric intermediates, whereas those of the second group seem to replicate by a protein-priming mechanism, similar to that of bacteriophage 29 (264). However, initiation of replication from an internal origin in a plasmid with a terminal protein has been reported (48). Linear plasmids have been reviewed previously (123), and they will not be discussed here. Replication of plasmids from gram-negative bacteria has been specifically addressed (168a).

Concerning their genetic structure, plasmids have an essential region which contains the genes or loci involved in replication and its control. The organization of this essential region corresponds, in general, to the one described in the replicon model. 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. Some of these so-called dispensable genes are involved in processes such as plasmid transfer and spread among bacteria, resistance to antibiotics and heavy metals, resistance to radiation, and transfer of DNA to higher eukaryotes. Within the plasmid essential region, several genes and sequences can be considered. (i) The first is the origin(s) of replication (generically termed ori), which is characteristic of each replicon. (ii) Although this is not a general feature, many plasmids encode a protein involved in the initiation of replication, usually termed Rep protein. (iii) The third is the plasmid-borne genes involved in the control of replication. 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. These specific sites are the hallmark of a class of replicons that are different from replicons that do not require specific initiators.

Replication by the theta-type mechanism has been most extensively studied among the prototype circular plasmids of gram-negative bacteria, although this replication mode has also been described for plasmids isolated from gram-positive bacteria, namely, the streptococcal/enterococcal Inc18 group (40), some lactococcal replicons (152), and at least one Bacillus subtilis plasmid (192). DNA replication through the theta mechanism involves melting of the parental strands, synthesis of a primer RNA (pRNA), and initiation of DNA synthesis by covalent extension of the pRNA (163). DNA synthesis is continuous on one of the strands (leading strand) and discontinuous on the other (lagging strand), although synthesis of the two strands seems to be coupled (reviewed in references 148 and 326). Theta-type DNA synthesis can start from one or from several origins, and replication can be either uni- or bidirectional. 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"). The replication intermediates can also be monitored by one- or two-dimensional electrophoresis. 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.

With some exceptions, plasmids using the theta mechanism of replication require a plasmid-encoded Rep initiator protein. Some replicons may require the host DNA polymerase I (DNA Pol I) during the early stages of leading-strand synthesis. Some features of various well-known replicons which are described here are depicted in Fig. 1.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Origins of replication and related regions of some representative theta-replicating plasmids from gram-negative bacteria. Plasmid names (left), origins of replication (center), and Rep initiator-binding sites (right) are indicated. The symbols used are as follows: solid boxed arrows correspond to repeated Rep-binding sequences (iterons); open arrows above maps indicate inverted repeats that have partial homology to the iterons; solid arrowheads indicate repeats found in AT-rich regions (A+T); for R1, arrows indicate the imperfect palindromes initially recognized by the RepA initiator protein. Promoters are indicated as open arrowheads below the maps. Other sites of interaction are as follows: IHF-binding sites (open rectangles), dnaA boxes (solid rectangles); FIS-binding sites (hexagons), dam methylation sites (solid circles), and primase assembly sites (pas). Other sites are indicated in the figure. 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).

(i) General features. With some exceptions, initiation of plasmid DNA replication requires a specific plasmid-encoded Rep initiator protein. This is reflected by the presence, at the origin of replication, of specific sequences with which the Rep protein interacts. Additional features found in many origins of theta-replicating plasmids are (i) an adjacent AT-rich region containing sequence repeats, where opening of the strands and assembly of host initiation factors occur, and (ii) one or more sites (dnaA boxes) where the host DnaA initiator protein binds (30, 163). Multiple Dam methylation sequences, which are present in the origin of replication of the Escherichia coli chromosome, oriC, can also be found at the origin of replication of plasmids such as P1 (36, 38) and pSC101 (30). Methylation is not essential for replication, its role being primarily in postreplication (3). Dam methylation sequences are not present in other plasmid replicons.

(ii) Iteron-containing origins. 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. 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). Among plasmids which restrict their establishment to a single or a few species of enterobacteria, iterons have been described for several replicons like P1 (5), F (209, 295), pSC101 (52), R6K (97, 98, 277, 278), Rts1 (144), and pColIV-K30 (247). 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). 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). Iterons can also be found outside the origin region in some plasmids (P1, F, RK2, R6K, Rts1, and pColIV-K30). These iterons, unlike the origin iterons, are not required for initiation but play an important role in the control of replication, as the origin iterons do. In plasmids that do not have auxiliary iterons, the origin iterons are the only locus involved in control (see below).

(a) Plasmid R1. Initiation of replication of R1 is dependent on a plasmid-encoded initiator protein, RepA. The minimal region required for RepA-dependent replication (oriR) is included within a 188-bp DNA region (183) and comprises (i) a 9-bp dnaA box, (ii) a contiguous 100-bp region where RepA interacts, and (iii) an adjacent AT-rich region containing three 9-mers. 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. This second site is contiguous to the AT-rich region. Sites 1 and 2 share a core sequence (g/tTTAAA) that is an imperfect palindrome (101). The intervening sequence between the sites shows potential intrinsic curvature. The presence of the dnaA box optimizes the action of the DnaA protein at the origin, both in vivo and in vitro, but it is not absolutely required for the DnaA-dependent replication of R1 (233). EM of replicating intermediates obtained in vivo and in vitro shows that initiation of R1 replication occurs in a locus that is separated from the minimal origin region (78). 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).

(b) Plasmid ColE1. ColE1 is the prototype of a class of small multicopy plasmids that replicate by a theta-type mechanism. Unlike R1, ColE1 does not require a plasmid initiator protein but requires DNA Pol I to initiate 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 first two sequences are the most relevant, since they are required for ColE1 replication in the presence or absence of DNA Pol I and RNase H (57, 158, 211). The origin of ColE1 replication, defined as the transition point between RNA II primer and DNA synthesized by DNA Pol I, has been positioned 555 bp downstream of the start point of RNA II (24, 300). This transition point corresponds with data obtained in vivo for plasmid pMB1 (closely related to ColE1) (29). Analysis of replication intermediates of ColE1 by EM, located a single origin and showed that replication is unidirectional. At an early stage, leading-strand synthesis proceeds in the absence of lagging-strand synthesis (297, 298).

(c) Plasmid pLS20. An interesting example of plasmid from gram-positive bacteria is the B. subtilis plasmid pLS20, for which a preliminary characterization has been reported (192). This plasmid replicates by the theta mechanism, and its replication is independent of DNA Pol I and of a Rep initiator protein. Several palindromes flanking a putative dnaA box are located within the origin of replication of pLS20.

Rep proteins. Up to now, dozens of plasmids have been isolated from most bacteria, but not many of them have had their basic replicons dissected and characterized to the level of their nucleotide sequence, and even fewer replicons have been genetically and biochemically studied in detail. The classic way of classifying plasmids is to distribute them among incompatibility groups, whose members have very similar origin sequences and replication control mechanisms. However, due to the difficulty to cope with a complex experimentally based classification of each newly isolated replicon, a criterion based on sequence comparisons appears to be much more practical. Such a criterion could be the comparison of the amino acid sequences of Rep initiator proteins, since they are encoded by most of the plasmids and they share common functions. Rep proteins recognize specific sequences at the origin of replication, similar to the DnaA initiator protein in bacterial chromosomal replication, and they generate a nucleoprotein initiation complex in which essential macromolecular interactions take place (Rep-DNA, Rep-Rep, and Rep with other initiation proteins of the host) (30). In addition, many Rep proteins can generate complexes that negatively regulate their synthesis and the frequency of initiation.

View larger version (99K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   FIG. 2.   Sequence alignment and phylogenetic tree of Rep initiator proteins from theta-replicating plasmids. (a) Sequence alignment of the Rep initiator proteins of nine related plasmids of the iteron-containing class. Sequences were aligned with the program CLUSTAL W (version 1.5) by using, for pairwise alignment, gap opening and extension penalties of 10.0 and 0.1, respectively, and the protein weight matrix BLOSUM30. For multiple alignment, the delay for divergent sequences was set to 40% (294). The degree of sequence identity to the pPS10 initiator sequence for conserved residues is shown: *, identical residues in eight or nine of the total sequences; , identical residues in five to seven sequences. 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). The two characteristic motifs found in the Rep initiators, LZ (hydrophobic heptad residues pointed to by open arrowheads) and HTH, are indicated. The EMBL database accession numbers are as follows: pPS10, X58896; pECB2, Y10829; pRO1614, L30112; pCM1, X86092; pFA3, M31727; pSC101, K00828; pCU1, Z11775; RepFIA, Y00547; R6K, M65025. (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). The sequences for 35 initiators were retrieved from databases, and a preliminary alignment was performed with CLUSTAL W (data not shown). 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). Distance matrixes were corrected for multiple substitutions by the method of Kimura. The phylogenetic tree was built up according to the UPGMA method with the program GROWTREE (95). For further discussion, see the text.

(i) Protein-protein interactions: the leucine zipper-like motif. A protein-protein interaction motif resembling the LZ is present in several plasmid-encoded Rep proteins. The LZ motif is responsible for dimerization in several eukaryotic regulatory proteins, through formation of two-stranded coiled coils (172). LZ-like motifs have been detected in the N-terminal region of the Rep proteins of several plasmids (103, 215) (Fig. 3). A mutational analysis has been carried out in the LZ-like motif of the RepA protein of plasmid pPS10 (94). Substitutions of the first two Leu residues of the LZ-like motif (d position according to a coiled-coil nomenclature) with Val resulted in a 13-fold decrease in the RepA association constant (as determined by sedimentation equilibrium analysis of maltose-binding protein-RepA fusions). This finding indicates that the LZ-like motif is a protein-protein interaction interface that regulates the equilibrium between monomers and dimers of the RepA protein. A conservative AlaVal change in a different residue of the motif (b position) has no effect on monomer-dimer equilibrium, which points to a relevant and specific role in dimerization for the Leu residues of the motif. 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. Further analyses indicated that RepA monomers bind to the iterons of the origin of replication whereas dimers of the protein bind to the repA promoter region, pointing to the functional relevance of the two forms of the RepA protein. Similar results have been obtained with the RepA protein of plasmid pSC101. This protein exists in a monomer-dimer equilibrium, although it is mainly in the monomeric form at the protein concentration present in cells harboring wild-type pSC101 (134). However, when the repA gene is overexpressed, replication is inhibited (133, 308). 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). 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.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   The theta-type replicon of the Pseudomonas plasmid pPS10. The iteron-containing origin (oriV) and motifs found in the replication initiator protein (RepA) are depicted. The minimal origin (oriV) of pPS10 plasmid is a good example for a "canonical" iteron-containing origin. It is composed of four contiguous and identical 22-bp iterons arranged as direct repeats (half arrows), flanked by a 9-bp dnaA box (hatched) and AT- and GC-rich sequences (215). The pPS10 replicon also contains the repA gene, encoding the RepA initiator protein (shadowed ovals). RepA is under a monomer-to-dimer equilibrium, which has consequences for protein activity: RepA dimers bind to an inverted repeat (with a sequence partially homologous to that of the iterons) that overlaps with the repA promoter (P), acting as self-repressor of repA expression, whereas RepA monomers bind to the iterons to form the initiation complex (94). Protein motifs found in RepA are depicted under the protein gene. The LZ motif, responsible for protein dimerization, is represented as a helical wheel projection, in which the hydrophobic spine of Leu residues and the chemical nature of the other displayed residues is indicated (103). For the HTH motif, involved in binding to DNA, the two proposed -helices are underlined and a stretch of basic residues at the C end of the DNA recognition helix is indicated (+), whereas an arrow points to the Gly residue thought to start the turn (93).

(ii) Specific binding of Rep proteins to DNA: the helix-turn-helix motif. As mentioned above, Rep proteins are able to specifically recognize DNA sequences in the origin region. In addition to this, some of the Rep proteins autoregulate their own synthesis at the transcriptional level by binding to sequences in the rep promoter (operator) which show some degree of homology to those present in the origin region. 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). rep mutants leading to impaired Rep protein-DNA interactions have been found in various plasmids.

(i) DNA replication dependent on plasmid initiators. Initiation of replication requires the assembly of the complete replication machinery including DNA polymerase III holoenzyme (DNA Pol III-HE), DnaB helicase, and primase at the plasmid origin. 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. Most of the theta-type replicons require, at least, a plasmid-encoded Rep protein and the host DnaA protein for the initiation step. The general organization of the origin region in these plasmids resembles the arrangement found in oriC (30). 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. The AT-rich repeats have also been involved in the transfer of the DnaB-DnaC complex to oriC. In the theta-replicating plasmids, the Rep protein binds to specific sequences in the origin, forming a nucleoprotein preinitiation complex analogous to the one formed by DnaA at oriC. 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. The structural organization of the initiation complex could be facilitated by host factors such as HU, IHF, or FIS. 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).

(a) Plasmid pSC101. RepA, the initiator protein of plasmid pSC101, exists in a monomer-dimer equilibrium, which determines the efficiency of RepA in replication (134). 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). However, interactions of the RepA dimers with the inverted repeat also play a role in replication in the absence of the par locus (197). Initiation of pSC101 replication requires, in addition to RepA (308), the DnaA host replication initiator (113), and IHF proteins (91, 283). Binding of IHF to its target, within the AT-rich region, leads to DNA bending (283), which promotes interactions between DnaA molecules bound to dnaA boxes separated by some 200 bp (282). Binding of RepA to the origin region further stabilizes DnaA contacts with the distant dnaA boxes (282). The RepA-DNA-DnaA complex plays a role in replication but also in partitioning of the plasmid (54). Stable plasmid inheritance requires the par locus, which is close to the origin region: this locus contains a site for DNA topoisomerase II and also determines the proper supercoiling at the origin region needed for initiation (53, 132).

(b) Plasmid P1. Plasmid P1 replication is dependent, both in vivo and in vitro, on the specific initiator protein RepA (6, 313) and on the host DnaA protein (110, 313). Formation of the initiation complex requires the monomeric form of RepA (315), and RepA-DNA binding is stimulated by heat shock chaperones. 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). However, growing evidence indicates that the chaperones are required to activate the monomers of RepA (50, 78a, 236). Binding of the activated RepA monomers to the five iterons of the origin results in wrapping of the DNA around RepA, presumably due to in-phase bending of DNA (206). RepA monomers contact each iteron through two consecutive major grooves on the same face of the DNA helix (242). RepA alone is unable to melt the origin; this role is performed or favored by DnaA, which also stimulates the DNA-binding activity of RepA (206). 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. Although either of the sets, or even just one dnaA box that conforms exactly to the consensus, is sufficient to support DnaA-dependent replication (4, 36, 38), melting of the origin region by DnaA is maximally efficient when both sets are present, probably due to DNA looping mediated by DnaA bound to the two sets (206). 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). Efficient replication of P1 requires adenine methylation of the five GATC sites of the origin. These GATC sites are clustered in direct heptamer repeats which are separated from the RepA-binding site by a GC-rich spacer (1, 2, 37).

(c) Plasmid RK2. Important information on the initial events of replication of plasmid RK2 has been obtained (161a). 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). This binding promotes, in the presence of HU protein, local strand opening within the AT-rich region of the origin. Interactions of the DnaA protein of the host with four DnaA boxes present in this region are also required for initiation of plasmid replication. These interactions increase, but are not strictly required for, the opening of the strands. 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. This suggests a role of TrfA in the repositioning and activation of the DnaB activity (79b). The requirement of particular DnaA boxes is host-dependent (79a). This is consistent with the plasticity of the RK2 origin with respect to structural requirements for replication in different bacterial hosts.

(d) Plasmid R6K. As stated above, replication from the  ori of R6K requires  protein (96, 160, 272, 278). This protein can recognize different types of DNA sequences: iterons, enhancer, inverted repeats in the promoter of the pir gene (encoding the  protein), and the AT-rich segment of the origin (174). The  initiator promotes the initiation of replication from three origins of replication: , , and  (reviewed in reference 87). ori- is in a central position, separated by 3 and 1.2 kb, respectively, from ori- and ori-. ori- contains seven 22-bp iterons, flanked by two IHF-binding sites and two dnaA boxes. Contiguous to the iterons is an AT-rich region which contains one of the dnaA boxes and one of the IHF-binding sites (62). ori- contains half an iteron, and ori- one complete iteron that are essential for function. Under standard conditions, ori- and ori- are more active than ori-, but they depend on a distant enhancer for activity (146, 147). This enhancer partially overlaps ori-, but its activity and the origin function have been distinguished by mutational analysis.

(e) Plasmid R1. Plasmid R1 is the most extensively studied member of the IncFII family of plasmids. In vivo and in vitro replication of R1 requires the initiator protein, RepA (77, 159, 183, 305). oriR, defined as the minimal region required for RepA-dependent replication of R1 in vitro, is bound specifically by RepA (101, 183, 184). Unlike the above cases, oriR does not contain iterons. RepA protein, probably as a dimer, recognizes sequentially (albeit with different affinities) the cores of two partially palindromic sequences (Fig. 4) (101). These sequences are located on the same face of the DNA helix, within a region of potential curvature, and are 8 helical turns apart (79). Interactions between RepA molecules bound to each of the sites could be responsible for DNA looping at the ends of a 100-bp region within oriR that is protected by RepA against DNase I cleavage (101). Following formation of the initial complex, additional RepA molecules could bind to the intermediate region by cooperative protein-protein interactions, generating a high-order complex. 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). The RepA-DNA complex seems to melt the DNA strands in the AT-rich region (185). In vitro replication of R1 requires DnaA protein (184, 231). 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). It is likely that RepA-DnaA contacts guide the entrance of the DnaA protein in oriR, because the dnaA box is not absolutely necessary for the DnaA-dependent replication of R1 (233). Surprisingly, although in vitro replication of R1 is dependent on DnaA, this protein is dispensable for the replication of IncFII plasmids in vivo (20, 290). In vivo replication in the absence of DnaA is inefficient, but plasmid copy mutants that increase the levels of RepA protein improve the efficiency of replication (20). These results show the essential role of RepA in origin activation and imply that RepA could promote melting of the DNA strands at the origin and loading of the DnaB-DnaC complexes.

View larger version (31K): [in this window] [in a new window]   FIG. 4.   RepA-oriR complexes in initiation of R1 plasmid replication. A 100-bp region in the oriR replication origin is continuously bound by the plasmid RepA protein to form the initiation complex. There are no iterons in oriR, but two partially palindromic 10-bp sequences (sites 1 and 2) are found at the ends of that 100-bp region. They are flanked, respectively, by a consensus dnaA box and by three AT-rich sequences. These three sequences are believed to be melted to allow the DnaBC complex access to the open origin. 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. 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. Binding of RepA dimers to sites 1 and 2 would generate a small DNA loop, held together by protein-protein interactions. The DNA loop would be filled afterwards with more RepA molecules that are brought to the complex mainly by protein-protein interactions. This model is based on experimental gel mobility shift assays and footprinting data with both wild-type and mutant RepA proteins and oriR sequences (101). Arrowheads indicate DNase I-hypersensitive sites (the size is proportional to the intensity of cleavage), whereas arrows point to strong cleavage sites for hydroxyl radicals. The interaction of DnaA host initiator with its DNA-binding site is dependent on the previous formation of the RepA-oriR nucleoprotein complex (233). A requirement for a DnaK chaperone has been described for R1 DNA replication (105). 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.

(f) Plasmids ColE2 and ColE3. As mentioned above, the smallest of all the prokaryotic origins described so far have been defined in the ColE2 and ColE3 replicons (322). These plasmids require for replication a plasmid-specific initiator, but, like ColE1 (see below), they also require DNA Pol I to initiate leading-strand synthesis. Initiation of ColE2 and ColE3 replication is dependent on the synthesis of specific primer by their Rep proteins (288). A single-strand initiation site (ssi) for the priming of DNA replication has been located near the ori of ColE2 (219).

(g) Plasmids of the pAM1 family. 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. A model based on the synthesis of a primer RNA catalyzed by the host RNA polymerase (RNAP), the specific cleavage of the primer at the origin region (perhaps mediated by the Rep protein), and the extension of the 3' end catalyzed by DNA Pol I has been proposed (41).

(ii) Replication independent of plasmid-encoded initiator proteins. The best-characterized replicon that is independent of plasmid-encoded initiator proteins is ColE1. Initiation of ColE1 replication involves the consecutive activities of RNAP, RNase H, DNA Pol I, and DNA Pol III-HE (reviewed in references 163, 182, 248, 280, and 301). Transcription mediated by the host RNAP is required to synthesize the preprimer for leading-strand synthesis. 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. This synthesis is initially carried out by DNA Pol I. Steric hindrance of the bulky DNA Pol III-HE by the folded RNA II (upstream of the RNA-to-DNA transition point) probably prevents extension of the primer by this polymerase (188). DNA Pol I synthesizes about 400 nucleotides of the leading strand, exposing, on the displaced strand, a primosome assembly site (pas). Once the primosome is assembled at the pas site, it translocates in the 5'3' direction, unwinding the helix and priming the discontinuous DNA synthesis. At this point, DNA Pol I is replaced by the highly processive DNA Pol III-HE. The switch between DNA Pol I and DNA Pol III-HE could be favored by helix-destabilizing proteins bound to the template of the leading strand, which has to be exposed by the DnaB helicase (discussed in reference 280). Leading-strand synthesis can occur uncoupled from lagging-strand synthesis, and DnaG, but not DnaB, is dispensable for this uncoupled synthesis (281). 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 mechanism of this arrest is unknown. These events determine the unidirectional pattern of ColE1 replication.

Termination of replication. The points at which theta-type replication terminates can be actively determined by molecular interactions at particular sequences. The first replication arrest sequence, ter, was identified in plasmid R6K as a barrier to the unidirectional replication initiated in either ori- or ori- of this plasmid. Replication starts then from the initial origin in the opposite direction and progresses to completion (177). The R6K terminus acts as a temporal barrier to replication initiated in other replicons (160). The nucleotide sequence of the ter region was determined, and the replication terminus of R6K was cloned (17, 18). The organization of this sequence as two separable and polar terminus sites was recognized and verified (130). The recognition of the essential features of the ter site allowed the identification of similar sites in plasmids of the IncFII (R1 and R100) and IncFI (repFIC) groups, as well as in the chromosome of E. coli. 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 identification of ter homologous sites in the chromosome of E. coli triggered replication termination studies in this bacteria and also in B. subtilis, where DNA-arresting sequences (IR-I and IR-II) and a dimeric protein that promotes the termination of DNA replication, RTP, have been identified (reviewed in references 14, 19, and 120). Unlike Tus, which acts as a monomer, RTP acts as a tetramer of two dimers (261, 262).

The best-known examples of plasmids replicating by the strand displacement mechanism are the promiscuous plasmids of the IncQ family, whose prototype is RSF1010. Members of this family require three plasmid-encoded proteins for initiation of DNA replication. These proteins promote initiation at a complex origin region, and replication then proceeds in either direction by a strand displacement mechanism (266; reviewed in reference 263).

Origins of replication. 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). This region also contains the origin of replication, as defined by EM analysis of replication intermediates obtained in vivo (59) and in vitro (266). 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). The origin extends further with a nonessential region and two small palindromic sequences containing the ssiA and ssiB sites located in opposite strands (Fig. 5a). Iterons are the RepC-binding sites (111, 112). The inverted repeats at the ssi sites could favor the formation of hairpins. In these hairpins, base complementarity in the upper part of the putative stem is essential for replication, while base complementarity and sequence specificity in the lower part of the stem are important for primer synthesis (195). The ssiA and ssiB sequences are specifically recognized by the plasmid-encoded RepB primase, which primes continuous replication from these sequences (111, 128, 129). Genetic analysis indicated that a single ssi, in an orientation that favors priming and chain elongation away from the iterons, is sufficient for RSF1010 replication (118). This organization suggests that the origin of replication of RSF1010 can be separated into three functional loci: the iterons, the ssiA region and the ssiB region. The iterons and the adjacent AT-rich region function as a duplex-opening region, and the ssiA and ssiB sites form a priming region (263).

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Replication of plasmid RSF1010 by the strand displacement mechanism. (a) Origin of replication and related regions. The interaction sites of RepB (inverted repeat [convergent arrows]) and of RepC (iterons [boxed arrows]) are indicated. GC- and AT-rich regions are also depicted. (b) Model for initiation of replication by the strand displacement mechanism in plasmid RSF1010 (266). Replication occurs with opposite polarities from two origins (ssiA and ssiB), which are independently used. Interactions between the plasmid-encoded proteins RepC and RepA are indicated. Priming is catalyzed by RepB' (not shown). Thin lines indicate newly synthesized DNA, with the direction of synthesis indicated by arrowheads. See the text for details.

Rep proteins. As indicated above, replication of RSF1010 is promoted by the joint activity of three plasmid-encoded proteins, RepA, RepB, and RepC, that have, respectively, 5'3' helicase, primase, and initiator activity (111, 263). The RepC protein, a dimer of 31-kDa subunits, interacts specifically with the iterons of the origin (111, 112) and probably with the RepA helicase, promoting the exposure of the ssi sites in a single-stranded DNA (ssDNA) configuration (129, 266, 289). The RepA protein is a hexamer of 30-kDa subunits, and it contains two activities: an ssDNA-dependent ATPase and a 5'3' DNA helicase. Expression of repB from two in-frame alternative start codons results in two polypeptides of 36 and 38 kDa, which correspond to two functional forms of the RepB primase: RepB and RepB' (111, 267). The 38-kDa RepB' was shown to be identical to the RSF1010-encoded MobA protein (involved in conjugative mobilization) (266).

Replication mechanism. Replication of RSF1010 DNA is independent of the host-encoded DnaA, DnaB, DnaC, and DnaG proteins, whose roles are played by the combined action of the plasmid-encoded RepA, RepB, and RepC proteins (90, 111, 265). The template for initiation of RSF1010 replication is supercoiled plasmid DNA (78, 266). DNA Pol III-HE and SSB are required for replication. Figure 5b outlines a model for initiation of RSF1010 replication, proposed by Scherzinger et al. (266). The first stage of this process involves the binding of the RepC protein to the iterons of the origin. 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. Subsequent translocation in the 5'3' direction of the RepA helicase bound to the L strand (the DNA strand which has the same sequence as the mRNAs coding for 10 of the 11 known RSF1010 proteins) (267) melts the duplex, exposing and activating the ssi sites. Alternatively, the interaction of RepC with the iterons could induce the opening of the duplex near the ssi sites. The exposure of the stem-loop structure in the ssi sites is probably required for the assembly of the RepB-primase to initiate replication (195). Initiation at either ssi site can occur independently, and replication proceeds continuously, with the RepA helicase facilitating displacement of the nonreplicated parental strand as a D loop. Continuous replication from each ssi signal in opposite directions would originate a double-stranded DNA theta-shaped structure in the overlapping region and two D loops beyond this region. 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. The RepA helicase of RSF1010 works in the 5'3' direction, which implies that it is working while bound to the displaced strand. The end products of the strand-displacement replication mechanism are ss-displaced circles and double-stranded supercoiled circles. The ssDNA molecules could correspond to either DNA strand and therefore could contain either the ssiA or ssiB sequences. These sequences are used to initiate synthesis of the complementary strand, which converts the ssDNA templates into double-stranded supercoiled circles. Therefore, double-stranded DNA (dsDNA) molecules, displaced single-stranded circular molecules, and partial double-stranded circles can be formed in this mode of replication.