M, DNA molecular weight marker; c(enzyme) = 100 μM; c(pDNA) = 20 nM. First lane of each reaction indicates zero point. Before loading the samples were pre-heated at 75°C for 20 min to avoid shifted zones. A. Reaction course of Eco31I-N334D. Aliquots were withdrawn at 2, 5, 10, 30 and 60 min. B. Reaction course of Eco31I-D311A. Aliquots were withdrawn at 1, 2, 3, 4 and 5 h.