Figure 3.

Chemical rescue of the nuclease activity of the catalytically inactive EndA His160A variant by imidazole. (A) Aliquots of the reaction mixture containing 20 nM EndA H160A and 15 ng/µl pBSK plasmid in triple buffer at pH 8.0 were taken at the time points indicated and the reaction products analyzed by 0.8% agarose gel electrophoresis. The analysis shows an increase of open circular (oc) and a decrease of supercoiled (sc) plasmid DNA over time. Plasmid DNA incubated for 20 min in the absence of EndA H160A and plasmid (C) were loaded as controls. (B) The experiment was repeated at different pH-values (pH 5.5–9.5) and the ratio of open circular to super-coiled plasmid DNA plotted against time (n = 3). EndA H160A showed nucleolytic activity at slightly basic pH values ranging from pH 7.5 to 8.5.