Plasmid Rolling-Circle Replication

The main features that characterize rolling-circle replication (RCR) (see Fig. 1A) derive from its singular initiation mechanism, which relies on the sequence-specific cleavage, at the nick site of the double-strand origin (dso), of one of the parental DNA strands by an initiator Rep protein. This cleavage generates a 3′-OH end that allows the host DNA polymerases to initiate the leading strand replication. Therefore, the RCR initiation circumvents the synthesis of a primer RNA that is required in all other modes of replication of circular double-stranded DNA (dsDNA). Elongation of the leading strand takes place as the parental double helix is unwound by a host DNA helicase and the cleaved nontemplate strand is covered with the single-stranded DNA binding protein. Since the nascent DNA is covalently attached to the parental DNA, termination of a round of leading-strand replication implies a new cleavage event at the reconstituted nick site. This reaction is assumed to be catalyzed by the same Rep molecule that carried out the initiation cleavage and remained bound to the 5′ end of the parental strand while traveling along with the replication fork. A trans-esterification then occurs that joins this 5′ end to the 3′ end generated in the termination cleavage, releasing the displaced parental strand as a circular single-stranded DNA (ssDNA). This replicative intermediate serves as the template for the synthesis of the lagging strand, which depends solely on host-encoded enzymes and is initiated from a highly structured region of the ssDNA, termed the single-strand origin (sso).