The Mechanism of Rolling Circle DNA Replication and the Roles of Initiator Protein RepD

Abstract

Rolling circle replication is used by plasmids, which carry antibiotic-resistance genes and are transferred between certain bacteria, thereby transmitting the resistance. In this mechanism the two strands are asynchronously copied: one simultaneously with unwinding the parent plasmid, the other strand initially forms a single-stranded circle. As well as polymerase and helicase (PcrA), which are recruited from the host, an initiator protein is also required: RepD in the work described here. This binds specifically to the double-stranded origin of replication and initiates unwinding by nicking one strand and forming a covalent bond with the 5′-end. This nicking opens up a short single-stranded region and so allows the helicase and polymerase to bind and begin unwinding and replication, respecively. After travelling all around the plasmid probably as a single protein complex, the proteins then reach the newly synthesised origin of replication, which provides the signal for termination. RepD then does a series of strand exchanges to close the two plasmid circles. We are using a combination of measurements with whole plasmids and oligonucleotide models to elucidate the series of events at each stage of the replication. In particular, by following individual processes in real time, we are able to describe the order of biochemical steps that enable this process to occur.