Rep68 and TrwC/Rep nick sc plasmid DNA containing binding and nicking sites.

(A) Schematic representation of the scDNA nicking assay. When incubating a sc plasmid with an endonuclease, the protein nicks specifically inducing relaxation of the plasmid (open circular, oc), which will result in slower migrating DNA species when visualised on an agarose gel. (B) His-Rep68 and His-TrwC/Rep-mediated scDNA nicking. 100 ng of the plasmid containing the target sequence indicated on top of the gels was incubated as described in Materials and Methods with either purified His-Rep68 (left panels) or His-TrwC/Rep chimera (right panels). Products were resolved on 1% agarose gels, subsequently stained with ethidium bromide. “-“: sc plasmid incubated in the reaction buffer and processed as described above, but in the absence of protein. sc and oc products are indicated with arrows. The oc/sc ratios of quantified DNA bands are indicated at the bottom of the panels. The presented gels are representative of 3 independent experiments (n = 3).