FIGURE 6.

Exonuclease cleavage rates of WT and mutant enzymes with template-primer containing terminal mismatch (33/14). The reaction conditions were identical to those used for ssDNA. The exonuclease activity was assayed using a 75 nm concentration of the indicated enzyme and 1 nm 33/14 mismatch template-primer. The reactions were quenched after different time intervals (note that the time of incubation for R821A and R882A/Y824A was increased to 60 and 90 s) by the addition of Sanger's stop dye, containing 95% formamide. The products were resolved on 16% polyacrylamide, 8 m urea gel (A). To determine the rates of degradation by the WT and mutant enzymes on one-mismatch template-primer, the amount of primer remaining was plotted as a function of time and fit to a single-exponential decay, using nonlinear regression (B). C shows the representative hyperbolic fit of the rates at various concentrations of WT and R822A/Y824A double mutant enzymes with one-mismatch DNA substrate. The rates at various enzyme concentrations were determined in a manner similar to that shown in B.