The Mechanism of Escherichia coli Deoxyribonucleic Acid Polymerase I

The interaction of Mn2+ and nucleotides with Escherichia coli DNA polymerase I has been examined by kinetic and magnetic resonance techniques. Electron paramagnetic resonance studies show that the enzyme binds Mn2+ at one tight binding active site (Kd ≤1 µm, Ka = 1.2 µm), four ± one intermediate sites (Kd = 29 µm), and 20 weak inhibitory sites (Kd = 830 µm; Ki = 600 µm). The effect of bound Mn2+ on the longitudinal relaxation rate of water protons is enhanced by factors of 9.5 (first site), 16.1 (second site), 7.1 (third to fifth sites), and 3.5 (6th to 19th sites). Comparison of kinetic and binding data suggests that the tight Mn2+ site and possibly the intermediate Mn2+ sites function in polymerization. From competition studies with EPR and proton relaxation rate, Mg2+ displaces Mn2+ from the tight and intermediate sites. dTMP and dTTP bind to the tight Mn2+ site with equal affinity (K3 = 100 µm) and decrease the enhancement by 37 and 23%, respectively. In contrast, at the intermediate Mn2+ sites, dTTP (K3 = 5 µm) binds more tightly by an order of magnitude than dTMP (K3 = 50 µm) but both nucleotides produce similar lowering (28%) of the enhancement factors. These results suggest the formation of multiple e-Mn-nucleotide bridge complexes through the α-phosphoryl group at both the tight and intermediate Mn2+ sites. Coordination of the α-phosphoryl group of dTTP by e-Mn would increase its susceptibility to nucleophilic attack, thus facilitating nucleotidyl transfer. In contrast, no evidence is obtained for a role of Mn2+ in the binding of DNA to the enzyme. The interaction of multiple molecules of dTTP with the enzyme would permit the prealignment of substrate molecules on the enzyme prior to polymerization.