Figure 1.

Mutant D148E exhibits high fidelity DNA cleavage. (A) HNH motif of KpnI. The KpnI monomer model is shown as a ribbon with helices as spirals and strands as arrows. The Mg²⁺ and Zn²⁺ ions are shown as spheres. The side chains of active site residues, D148, H149 and Q175, are shown as sticks and labeled. (B) Effect of D148E mutation of KpnI on DNA cleavage specificity. Plasmid DNA pUC18 (14 nM) was incubated with various concentrations (25, 50 and 100 nM) of WT or mutant enzyme for 1 h at 37°C in the presence of 5 mM Mg²⁺ and analyzed on 1% agarose gel. C indicates DNA cleavage reaction in the absence of enzyme. Nc, L and Sc indicate the positions of nicked circular, linear and supercoiled forms of the plasmid, respectively. The asterisk denotes the promiscuous DNA cleavage products.