Steady-state kinetic analyses

A 5′-³²P-labeled primer annealed with template was extended in the presence of increasing concentrations of a single dNTP. The sequences of oligonucleotides (unmodified and BP-dG modified) used for steady-state kinetic analyses are shown in Fig. 2B,C. To ensure single-hit polymerization conditions (less than 20% of the primer is extended), polκ concentrations, the nucleotide concentration range and time of reaction were adjusted for every experiment (See Fig. ^S6 for more details). The reactions were incubated at 37 °C using the standard DNA-polymerase assay described above, and the reaction products were separated by electrophoresis on a 20% polyacrylamide gel containing 8 M urea. The percentage of primers extended by the polymerase was calculated using Storm ImageQuant software. The rate of product formation (ʋ, nMmin-1) was plotted as a function of dNTP concentration, and the data were fit by a nonlinear regression curve to the Michaelis–Menten equation using GraphPad Prism software (GraphPad, San Diego, CA). V_max and K_m values were obtained from the fitted curves, and k_cat was calculated by dividing the V_max by the enzyme concentration.