Figure 1.12 Theta model of plasmid replication. (a) The structure of the origin of replication in the broad‐host‐range, single‐copy, IncP plasmid RK2, showing the relative positions and numbers of the DnaA boxes, the iterons (binding sites for the replication protein, TrfA), the AT‐rich DNA unwinding element (DUE), and the adjacent GC‐rich sequence. (b) A replication cycle is shown for an idealised plasmid using theta replication. Replication begins with the binding of the replication protein to the iterons and the recruitment of the host‐encoded DnaA to the DnaA boxes. (c) The DNA in the DUE becomes single‐stranded, creating a replication bubble to which host‐encoded replication proteins are recruited. (d, e) Depending on the plasmid, DNA synthesis proceeds either uni‐ (e.g. ColE1) or bidirectionally (e.g. RK2). (f) The products of plasmid replication are catenated, double‐stranded circles and these are unlinked by Topo IV. (g, h) The unlinked plasmids are negatively supercoiled by DNA gyrase, recreating the substrate for another round of replication.

Iterons are the binding sites for plasmid‐encoded Rep (replication) proteins. The Rep proteins are needed to initiate plasmid DNA replication, but they can also inhibit this process. In addition, Rep proteins have roles as transcription regulators, acting as auto‐repressors. They are also subject to turnover by host‐encoded proteases (Konieczny et al. 2014). The positions, numbers, orientations, lengths, DNA helical phasing, and spacer lengths of iterons vary from plasmid to plasmid and making alterations to any of these details within a particular plasmid typically has a negative effect on replication (Konieczny et al. 2014). In RK2, the Rep protein (called TrfA) and DnaA both target the origin, which has an A+T‐rich DUE adjacent to the iteron arrays. Some iteron plasmids (but not RK2, which requires HU) have a requirement for IHF binding to the origin for efficient initiation of replication (Shah et al. 1995). Sites for Dam methylation (5′‐GATC‐3′) and SeqA binding are also features of some iteron‐dependent origins (Brendler et al. 1995). SeqA binding blocks replication initiation by excluding the replication proteins. A GC‐rich sequence motif is located adjacent to the DUE in RK2, but its significance is unclear (Figure 1.12). Other iteron origins have requirements for the FIS NAP and the IciA protein, an inhibitor of DNA unwinding at the DUE.

      Many of the cis or trans‐acting components of iteron origins, and their architectures, are reminiscent of oriC on the chromosome. The TrfA replication protein of RK2 interacts with, and recruits, the DnaB helicase. The ability of a plasmid replication protein to recruit a host helicase may be a determining factor limiting plasmid host range (Zhong et al. 2005). TrfA also acts with Hda, the inhibitor of DnaA activity, to prevent over‐initiation of RK2 replication (Kim et al. 2003). It has been suggested that TrfA has a motif that is suitable for interaction with the β‐clamp of E. coli DNA Pol III (Kongsuwan et al. 2006). In addition to DnaB, iteron‐based initiation also requires DnaC (in E. coli), the DnaG primase, DNA gyrase, the Pol III holoenzyme, and the SSB, as is also seen at oriC (Section 1.3). Unlike initiation of chromosome replication at oriC, initiation of plasmid DNA replication at iteron origins is ATP‐independent; there is no requirement for the DnaA‐ATP form of DnaA (Konieczny et al. 2014).

An important mechanism of copy number control in iteron plasmids is ‘handcuffing’, where the monomeric Rep proteins bound to iterons on two plasmids dimerise, bridging the two replicons (Das and Chattoraj 2004). Handcuffing may be counteracted by the DnaJ‐DnaK‐GrpE protein chaperone triad, which can convert the Rep dimers to monomers (Toukdarian and Helinski 1998). The Rep proteins downregulate replication initiation through the auto‐repression of their own genes (Kelley and Bastia 1985). The level of active Rep proteins in the cell is controlled by proteolysis and protein chaperones: monomers are active and dimers are inactive in promoting replication (Konieczny et al. 2014). Active Rep proteins are a proxy for the number of plasmid copies in the cell and allow feedback through dimer formation to downregulate Rep activity, and hence to inhibit the synthesis of new plasmid copies. Antisense RNAs also play important roles in modulating plasmid replication through their ability to interfere with the expression of plasmid‐encoded replication factors (Brantl 2014).

      RK2 and its plasmid relatives use a theta model of DNA replication (Figure 1.12). Plasmid ColE1, the backbone for many cloning vectors used in recombinant DNA technology, also uses theta replication, but differs from RK2‐like plasmids in relying on host factors to open the double‐stranded origin and to prime synthesis (Lilly and Camps 2015; Wang et al. 2004). DNA duplex unwinding is driven by transcription of a stable RNA pre‐primer that forms an R‐loop in the ori region of ColE1. This process is driven by negative supercoiling of the plasmid DNA. RNase H then processes the bound RNA to generate the primer RNA that is then extended by Pol I. This marks the beginning of leading strand DNA synthesis. As the newly synthesised DNA strand makes progress through the plasmid DNA duplex, it base pairs with the template to create a D‐loop that recruits PriA. Pol III takes over leading strand synthesis and initiates the synthesis of the lagging strand; the converging replisomes continue moving until they are at or near the termination site terH (Nakasu and Tomizawa 1992). Gaps between the strands are then filled in by Pol I (Troll et al. 2014).

Rolling circle replication allows plasmids to replicate independently of chromosomal DNA (Khan 2005). The process relies on a nick made by a plasmid‐encoded initiator protein in one plasmid DNA strand, providing a primer for leading strand initiation and a lagging strand origin (Figure