Figure 1.

Overview of Rep mediated rolling circle replication. (A)(1) Rep protein binds to a hairpin loop at the origin of replication and nicks the (+) strand (red), forming a covalent adduct with the DNA. (2) The Rep protein recruits PcrA helicase to the nick site and following strand separation DNA synthesis commences from the 3′-end of the nick. (3) Synthesis of a new (+) strand (green) displaces the original (+) strand (blue) and on completion the Rep protein nicks the newly synthesized origin and religates the ends of the displaced strand, producing a single stranded product. A new (−) strand is synthesized by the host cell replication machinery. (4) Replication continues 9–11 bases (magenta) beyond the nick site to regenerate the hairpin loop which the Rep protein nicks via one active site while religating the ends of the newly synthesized (+) strand with the other completing synthesis. The 11 base-pair oligonucleotide remains covalently attached to the Rep protein to produce a catalytically inactive Rep* molecule. (B) The DNA sequence at the origin of replication of S. aureus plasmid pC221. The two inverted repeats are ICRII, which forms a conserved stem–loop structure presenting the nick site at the tip in the (+) strand, and ICRIII, a plasmid-specific repeat which spans the Rep protein binding region and permits discrimination of cognate plasmids by their Rep proteins in vivo (highlighted green).