Initiation of Asymmetric Rolling-Circle Plasmid Replication by RepD Studied using Magnetic Tweezers

Abstract

Bacterial plasmids, like the ones of T181 family, are replicated by an asymmetric rolling-circle mechanism. The replication is initiated by the protein, RepD, which binds to the origin of replication (oriD) on the plasmid and makes a single strand nick forming a covalent complex with the DNA “plus-strand”. This enables the helicase, PcrA and the DNA polymerase, Pol-III to bind the short length of exposed single-stranded DNA allowing replication of the “minus-strand” to commence. In the final stage of DNA replication, RepD terminates the process by religating the two ends of the plus-strand leaving behind the, newly formed, double-stranded daughter plasmid and the circular plus-strand, which is later replicated by a different mechanism. We used a custom-built magnetic tweezers device to observe initiation (nicking) by RepD. The initiation site, oriD, consists of three inverted complementary repeats that are predicted to form secondary DNA structures (hairpins). When the circular plasmid is damage-free DNA gyrase converts the relaxed circular DNA into a compact, negatively supercoiled, form which favours extrusion of secondary structure motifs. Using magnetic tweezers to artificially supercoil a length of DNA containing the oriD sequence, we found RepD nicking activity is highly sensitive to the degree and sign of supercoiling. Positive supercoiling (over-winding) strongly inhibits nicking whereas negative supercoiling stimulates nicking. We propose that DNA supercoiling acts as a gate-keeper to ensure plasmids are damage-free before DNA replication is initiated and the time required for daughter plasmid supercoiling may regulate plasmid copy number. To corroborate this idea we used magnetic tweezers to directly observe formation of DNA secondary structure in the absence of RepD and we have preliminary results showing how the structures change in the presence of RepD.