DNA Unwinding by PcrA Helicase and RepD using TIRF and Magnetic Tweezers

Abstract

Bacterial plasmids of the pT181 family are replicated by an asymmetric rolling-circle mechanism. We have studied plasmid replication that is initiated when the protein, RepD, binds to the origin of replication (OriD) making a single strand nick and forming a covalent complex with the DNA. This exposes a short length of single-stranded DNA (ssDNA) allowing PcrA helicase to bind and start unwinding the plasmid. While the DNA is being unwound, DNA Polymerase III travels behind PcrA and synthesizes a new, complementary, strand. The other strand becomes decorated with single-stranded DNA binding protein (SSB) and is later replicated by a different mechanism. In earlier studies, we monitored individual PcrA unwinding reactions using TIRF microscopy to image the accumulation of fluorescently labelled SSB on the nascent ssDNA strands (Fili et al., (2010) Nucl Acids Res. 38:4448-4457). We are now using a custom-built magnetic tweezers device to observe initiation (nicking) by RepD and translocation by PcrA. Our initial studies have focussed on the effect of DNA supercoiling upon RepD DNA-nicking kinetics and on DNA unwinding by PcrA. 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 (e.g. no single strand breaks) DNA gyrase converts the relaxed open circle into a compact, negatively supercoiled, form which favors extrusion of secondary structure motifs. Using magnetic tweezers to artificially supercoil a length of DNA containing the OriD sequence, we found that RepD nicking is highly sensitive to DNA supercoiling and negative supercoiling serves as a gate-keeper to ensure plasmids are damage-free before DNA replication is initiated.