Figure 1.

Role of replication initiation proteins in initiation in class A theta plasmid replication (based on references 43 and 97). Class A theta replicons typically include a replication initiator gene: rep (rectangle), DnaA boxes upstream or downstream of the Rep (square), iterons (arrow heads), and an AT-rich DNA unwinding element (DUE, oval). Sites for binding of nucleoid associated-proteins (such as IHF) or for methylation by Dam methylase are also frequently present as well but not shown. oriV of the RK2 plasmid (which belongs to the IncP compatibility group) is shown as an example. RK2’s replication initiator is TrfA. The trfA gene is under the control of a strong promoter. Transcription produces two products (a longer and a shorter one) that in this figure are considered largely redundant. During plasmid origin recognition (phase 1), replication initiation proteins (in their monomeric forms) cooperatively bind iterons until those iterons are saturated. The formation of an open complex (phase 2) involves unwinding of the DUE and continued binding of the replication initiator into the bottom strand of ssDNA; the rest of the bottom strand of ssDNA and the top strand are bound by SSB, which is a tetramer. In the case of RK2, the plasmid encodes its own SSB, which is under the control of the same promoter as the replication initiator gene (trfA). DnaA enhances/stabilizes the formation of the TrfA-mediated open complex and assists with the recruitment of DnaBC. Finally, the longer form of TrfA also assists in strand-specific replisome assembly in a DnaA-independent manner via direct interaction with the β clamp and through a sequence-specific interaction with one strand of the plasmid origin DUE (8, 136).