Figure 3.
Uracil excision assays for product inhibition. Uracil excision assay using ∼0.1 pmol 5΄ end 32P-labelled ssU9 substrate with the indicated amounts of EcoUng or BdiUng in the presence of varying concentrations of uracil (i and ii), and ssF9 (a stable AP DNA mimic harbouring tetrahydrofuran at position 9) (iii and iv) were carried out as described in Materials and Methods. Reactions were resolved on 8M urea PAGE (15%) and imaged. The estimated values (%) of substrate (S) to product (P) conversion [P/(S + P) × 100] are plotted against the concentrations of the inhibitor in the reaction. The data were fitted with the one site-total of saturation binding equations (Y = Bmax * X/(Kd + X) + NS*X + background) (where Y is specific binding/activity, Bmax is the maximum specific binding/activity in the same units as Y, Kd is the equilibrium binding constant in the same units as X, NS is the slope of nonspecific binding in Y unit divided by X unit and background is the amount of nonspecific binding) using GraphPad Prism software. Amounts of product formed in control (without free uracil/AP DNA) reaction in panels i and iii were scaled to 100%, and the remaining activities shown relative to this reference to generate plots in panels ii and iv.