Rolling circle replication overview

Replication begins when the Rep protein, which is encoded on the plasmid (ORF A), recognizes a specific site on the plasmid (double-strand origin, or DSO) and catalyzes the nicking of one DNA strand. The Rep protein remains bound to the 5′ phosphate after the nicking action. The newly released 3′ hydroxyl on the opposite end serves as a primer for DNA synthesis. The host DNA polymerase uses the unnicked circular strand as a template, so that a single replication fork moves around a plasmid until it regenerates the DSO. A second copy of Rep protein catalyzes the cleavage of the newly formed DSO, effectively releasing a single stranded copy of the plasmid. In the absence of Rep, the replication fork continues to move around the template, forming a single stranded concatemer. The single strand origin (SSO), a non-coding element that forms extensive secondary structure, is required for synthesis of the lagging strand. SSO sequences vary considerably among different RCR plasmids, but are extremely important for robust replication of the plasmid in the cell [10]. Here we describe the engineering of the pWV01 RCR origin to create pBAV1K-T5, a very broad-host range expression vector.