In the assay for nicking supercoiled plasmid DNA, reaction mixtures containing 0.2 mmol MobA89K, MobA89KN258 or MobC89K protein, and 300 ng pMD-oriT plasmid substrate DNA were incubated in a total volume of 20 ml containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA and 2 mg ml -1 BSA (Pansegrau and Lanka, 1996) . After incubation overnight at 37°C, reactions were stopped by adjusting the mixture to 1% SDS and 0.1 mg ml -1 proteinase K, and the incubation was continued at 37°C for 30 min. The reaction mixtures were then subjected to electrophoresis analysis. Gel images were captured digitally, and band intensities were quantified with Quantity One software (Bio-Rad). The yield of nicked OC DNA was calculated after correction for the relative fluorescence of OC to CCC plasmid DNA.