Figure 4:

Equilibrium and kinetics of the fluorescence changes following incorrect nucleotide binding. (A) Equilibrium titration data follow a hyperbolic curve with a K_d of 130 ± 0.8 μM. (B) Stopped-flow fluorescence traces corresponding to the binding of dGTP at concentrations ranging from 25 to 1500 μM. The data could be fitted globally with three isomerization steps with forward/ reverse rates of 220/420, 30/100, and 12/7, respectively, but this fit is not unique and is not shown. Rather, the minimal model with a single isomerization is shown in Scheme 1. (C) Rate of nucleotide release from a mismatched ternary complex was measured by chasing the release of the mismatch with the correct nucleotide. The E•DNA_dd•dGTP complex (200 nM) was formed with 250 μM dGTP and then mixed with 2 mM dCTP. The fluorescence transition shows a single-exponential decay with a rate of 372 ± 5.4 s⁻¹.