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.
Strand Displacement Replication
The best-known examples of plasmids replicating by the strand displacement mechanism are the promiscuous plasmids of the IncQ family, whose prototype is RSF1010.
promiscuous 난잡한
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).
bidirectional 양방향성의
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).
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, 59339 helicase, primase, and initiator activity (111, 263).
respectively 각각
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 singlestranded 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 59339 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 RepB9 (111, 267). The 38-kDa RepB9 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 59339 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 doublestranded 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 59339 direction, which implies that it is working while bound to the displaced strand. The end products of the stranddisplacement 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 doublestranded 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.
Synopsis.
IncQ plasmids (typically RSF1010) are replicons that can be propagated in many different hosts. Replication of RSF1010 occurs from two symmetrical and adjacent singlestranded origins (ssiA and ssiB) positioned one on each DNA strand. Replication starts when these origins are exposed as single-stranded regions. 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. RepC recognizes directly repeated sequences of the origin adjacent to the AT-rich region, and RepA is a DNA helicase. Priming of DNA synthesis at these origins is catalyzed by the plasmid-specific primase (RepB). Synthesis of each one of the strands occurs continuously and results in the displacement of the complementary strand. Replication of this displaced strand is initiated at the exposed ssi origin. Due to the activities of the three plasmid replication proteins (RepA, RepB, and RepC), initiation of RSF1010 replication is independent of transcription by host RNAP and of host replication factors acting at the early replication stages (DnaA, DnaB, DnaC, and DnaG). This independence may account for the broad-host-range character of the IncQ replicons.
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