Fig. 2.
Single nucleotide incorporation in the presence of O-TIBO. (A) RT (50 nM), (5′−32P)-labeled 25·45-bp duplex DNA (100 nM), and O-TIBO were first incubated for a minimum of 20 minutes in buffer solution (50 mM tris-Cl, 50 mM NaCI, pH 7.5). This E·DNA solution was then rapidly mixed with 20 K.M dATP in buffer containing 10 mM MgCl2. The reactions were quenched with 0.5 M EDTA, pH 8.0 at the indicated times. The O-TIBO concentrations were 0 (○), 1.0 (●), 2.0 (□), 4.0 (■), 10.0 (△), and 20.0 (▲) μM. The concentrations are the final concentrations after 1 :1 mixing in the instrument, except for O-TIBO. The O-TIBO concentrations are those of the inhibitor in the initial E·DNA solution where equilibrium is established. The individual time courses were fit to a burst equation, y = A·(1 − exp(−kt) + m·t). (B) The amplitudes of the burst phases (●) were plotted against O-TIBO concentration and fit to a hyperbolic function (solid line) to yield a Kd value of 3.0 ± 0.2 μM.