In another set of experiments, linearized plasmid DNA was used as the substrate for the cleavage reaction. pMD-oriT plasmid DNA was linearized with an appropriate restriction endonuclease (NdeI or ScaI) that cleaves the plasmid only once before co-incubation with the proteins of interest. The resulting ssDNA fragments were separated on 0.9% alkaline agarose gels, and the amount of cleaved substrate DNA was determined.