Replication and Control of Circular Bacterial Plasmids - Rolling-Circle Replication

Rolling-Circle Replication

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). 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. RC replication was originally thought to be limited to ssDNA coliphages and to small multicopy plasmids isolated from gram-positive bacteria. However, there are known instances of plasmids isolated from gram-negative bacteria, from cyanobacteria, and from species of Archaea that use the RC mode for replication (83, 99, 127, 156, 324, 331). 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. 

For example, small plasmids like pRJF1 (2.6 kb) and pWV02 (3.8 kb), isolated from gram-positive bacteria, replicate by the theta mode (115, 152). Studies on the molecular mechanisms underlying RC replication have been done mainly with the staphylococcal plasmids pT181 (228), pC221 (292), pUB110, and pC194 (108) and with the streptococcal plasmid pMV158 and its Dmob derivative pLS1 (69). The current model for RC replication involves several experimentally distinguishable stages (Fig. 6). Replication is initiated by the plasmid-encoded Rep protein, which introduces a site-specific nick on the plus strand, at a region termed doublestranded origin (dso). The nick leaves a 39-OH end that is used as a primer for leading-strand synthesis, which most probably involves host replication proteins (at least DNA Pol III, SSB, and a helicase). Elongation from the 39-OH end, accompanied by the displacement of the parental plus strand, continues until the replisome reaches the reconstituted dso, where a DNA strand transfer reaction(s) takes place to terminate leadingstrand replication (see below). Thus, the end products of leading-strand replication are a dsDNA molecule constituted by the parental minus strand and the newly synthesized plus strand, and a ssDNA intermediate which corresponds to the parental plus strand. Unlike replication by the strand displacement mechanism, the ssDNA intermediates generated by the RC replication mode correspond to only one of the plasmid DNA strands. The pioneering work in Ehrlich’s laboratory showed that generation of ssDNA is the hallmark of plasmids replicating by the RC mechanism (108, 291). Finally, the parental plus strand is converted into dsDNA forms by host proteins initiating at the single-strand origin (sso), which is physically distant from the dso. The last step would be the supercoiling of the replication products by the host DNA gyrase.

 

Origins of leading-strand synthesis.

RC-replicating plasmids are made up of interchangeable gene modules (253). There is only one essential module. This harbors the functions for plasmid replication and includes the dso, the rep gene, and the plasmid elements involved in replication control. This module has been named the leading-strand initiation and control (LIC) region (69). In addition, plasmids may include an antibiotic resistance determinant, a gene involved in conjugative mobilization (mob), and one or two sso regions. Based on the homologies observed in the essential LIC module, four main plasmid families have been defined, their prototypes being pT181, pC194, pMV158, and the staphylococcal plasmid pSN2 (69, 84, 108, 228). Little information is available on the fourth plasmid family, and newly described plasmids from Pyrococcus abyssi and from extreme halophiles seem to belong to one of these families or to fall within a different, less well characterized, fifth family (83, 127). An interesting replicon is the natural phage-plasmid hybrid replicon, termed phasyl, which has been isolated from E. coli. It contains two functional origins: one has homology to the viral and complementary origins of the ssDNA coliphage M13, whereas the other origin requires activation by the phasyl-encoded Arp protein. Iterons or dnaA boxes typical of the theta-type replicons have not been found in phasyl (271). Two loci have been defined within the dso, namely, the bind and nic regions (70). The former locus includes the sequences needed for the Rep protein to bind to the plasmid DNA, whereas the latter contains the site where the Rep protein introduces the initial nick (Fig. 7). The two loci can be contiguous (pT181 family) or can be separated by up to about 100 bp (pMV158 family). A typical feature of the RC-replicating plasmids is that the nic regions are highly conserved among replicons of the same family whereas differences are found at the bind loci. This fact shows that Rep proteins of plasmids from the same family must have a common catalytic domain for phosphodiester bond cleavage and sealing whereas replicon specificity (i.e., Rep binding to the bind region) would be located in a different, nonconserved domain (see below). The DNA sequences of the bind regions are either an inverted repeat contiguous to the nick site (IR-III in the pT181 and pC194 families [Fig. 7]) or a set of two or three direct repeats separated from the nick site by intervening sequences (pMV158 family [Fig. 7]). The essential regions of the pT181- dso (IR-II and IR-III) involved in the interaction with the plasmid RepC initiator protein have been defined in a systematic study (312). IR-II (nic region) contains the RepC nick site, and the IR-III (bind region) is contiguous to it (Fig. 7 and 8). Whereas IR-II is conserved among plasmids of the pT181 family, IR-III is not, suggesting that the origin specificity is provided by IR-III. The proximal half of IR-III is more important for RepC recognition of the dso than is the distal half. In addition, the spacing and phasing between IR-II and IR-III are important for dso function (312). A similar picture is found in plasmids of the pC194 family. Conservation of the nic locus and divergence in the bind region can also be observed in the plasmids of the pMV158 family. In these plasmids, the distance between the conserved nick site and the direct repeats (the nonconserved bind locus) ranges between 14 and 95 nucleotides. The RepB protein of pMV158 binds in vitro to a dsDNA fragment containing the direct repeats (60). However, unlike the plasmids with iteron-containing origins described above, the pMV158 direct repeats do not constitute an incompatibility determinant toward pMV158 (70). 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). The nic region contains inverted repeats able to generate one or two hairpin structures (65, 216, 254). Three different types of nic regions, belonging to (i) the pT181 family, similar to that of M13, (ii) the pC194 family, exhibiting similarities to fX174, and (iii) the pMV158 family, with no relevant homologies to nic regions from known ssDNA coliphages, have been reported (Table 1) (69, 108, 228). The development of a system in which DNA strand discontinuities can be mapped with nucleotide resolution in vivo has allowed the identification of the nick sites of other plasmids of the pMV158 family, namely, pE194, and pFX2 (106, 328). The DNA sequences recognized by the Rep proteins to introduce the nick would be located on unpaired regions within these hairpins (IR-II in pT181; hairpin I in pMV158 [Fig. 7]), which accounts for the absolute requirement of supercoiled DNA as the substrate for replication (64, 201, 216). Genetic analysis pointed to the existence of a stemloop structure within the nic region of pC194 (196). However, for the related plasmid pUB110, no transient hairpins seem to be required to initiate replication (10). In vitro analyses have shown that IR-II of plasmid pC221 (closely related to pT181) or hairpin I of pMV158 are sufficient for the nicking-closing activity of their respective Rep proteins. This reaction is not plasmid specific, since cognate nic regions are also cross-recognized by these Rep proteins (201, 292, 332). In addition, in vitro replication of plasmid pC221 is reduced if a competing pT181-dso is present (332). In spite of such an in vitro recognition and extensive homologies of the Rep proteins and the dso of pT181 and pC221, there is no cross-reactivity between the Rep proteins and the dso of these plasmids in vivo, unless the Rep proteins are overexpressed (135).