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. 2015 Apr 9;6:135. doi: 10.3389/fgene.2015.00135

FIGURE 2.

FIGURE 2

Kinetic analysis of S305R and P1073L mutant. (A) Active site titration of S305R mutant. The S305R mutant (100 nM) was preincubated with 350 nM DNA template (25-mer/45-mer) for 10 min on ice. The binary complex was then rapidly mixed with 10 mM Mg2+ and 50 μM dATP (correct incoming nucleotide). The reaction was allowed to proceed for variable times before quenched by 250 mM EDTA. The product was resolved on a 15% denaturing polyacrylamide gel as shown in the upper panel, quantified and plotted against the reaction time in the lower panel. The data were fit to a burst equation as indicated in the text. (B) Kinetic incorporation of dATP for S305R mutant and P1073L mutant (C). For each concentration series, a preformed enzyme-DNA complex ([enzyme] > [DNA]) was mixed with variable concentrations of nucleotide (0.05, 0.2, 0.4, 1, 6, and 17 μM) for S305R mutant (B) and (0.2, 0.5, 1.5, 3, 5, and 10 μM) for P1073L mutant (C), respectively, and then quenched with 250 mM EDTA. Some of data shown in (B) were collected from (Qian et al., 2014). In each panel, the smooth lines represent the best fit to the model shown in Scheme 1 derived using KinTek Explorer software. The rate constants were determined and shown in . The error analysis of data collected for the S305R mutant was shown in Table 1. (D) DNA concentration-dependent kinetic incorporation of dATP for S305 mutant. The enzyme (55 nM) was preincubated with variable concentration of DNA substrate (10, 30, 50, 100, 200, and 300 nM) and was mixed with 100 μM dATP and 12.5 mM Mg2+ for varying time as indicated in the figure. (E) Excision of DNA containing a single T–T mismatch at the 3′-terminal of the primer. Enzyme (100 nM) was preincubated with 75 nM DNA substrate (25-mer/45-mer), and Mg2+ and excess unlabeled DNA were added to initiate the cleavage reaction. The remaining 25-mer was plotted against time and fit to a single exponential to yield an excision rate of 0.34 ± 0.04 s-1 for wild-type enzyme, 0.11 ± 0.02 s-1 for S305R mutant, and 0.28 ± 0.03 s-1 for P1073L mutant.