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. 2011 Feb 28;286(16):14480–14492. doi: 10.1074/jbc.M111.218750

FIGURE 5.

FIGURE 5.

KF-DNA complexes formed in the presence of noncomplementary dNTPs or rNTPs. An equimolar mixture of all three noncomplementary dNTPs or rNTPs (each present at the indicated concentrations) was added to the nanopore cis chamber with KF and the primer/template substrate shown in Fig. 3A each present at 1 μm. The templating residue at position N = 0 is dCMP. A, the effect of an equimolar mixture of dATP, dCTP, and dTTP on KF-DNA complexes captured at 180-mV applied potential on mean dwell time (i) and the fraction of captured DNA molecules that were KF-bound (ii). The total number of capture events for each of the concentrations of noncomplementary dNTPs from which the KF-bound fractions were determined was as follows: 0 μm, 2003; 50 μm, 829; 200 μm, 907; 600 μm, 1364; 800 μm, 1667; 1000 μm, 2285; and 1200 μm, 1998. iii, 1/(enzyme bound fraction) versus (incorrect dNTP concentration) and the linear fitting, which yields the binding affinity of incorrect dNTPs (the estimated value with standard error is described in the text). B, the effect of applied voltage on the mean EBS dwell time of KF-DNA complexes in the presence of a 0 (red circles) or 800 μm (blue circles) concentration of each of dATP, dCTP, and dTTP. C and D, noncomplementary nucleotide concentration dependence of EBS dwell times for KF-DNA complexes. C, mean dwell times on the nanopore at 40-mV applied potential for KF-DNA complexes in the presence of an equimolar mixture of dATP, dCTP, and dTTP (blue circles) or rATP, rCTP, and rUTP (red circles) at the indicated concentrations. D, mean dwell times on the nanopore at 40-mV applied potential for KF-DNA complexes in the presence of an equimolar mixture of dATP, dCTP, and dGTP (blue circles) or rATP, rCTP, and rGTP (red circles) at the indicated concentrations. In this panel, the residue at position N = 0 of the template strand is dAMP; this DNA substrate is otherwise identical to the substrate used in A–C. In C and D, the solid and dotted gray lines indicate the mean dwell time for the binary complex of KF and DNA and its 95% confidence interval, respectively. In all panels, error bars indicate the 95% confidence interval. Measurements at voltages lower than 180 mV (B–D) were made using the method illustrated in supplemental Fig. S4 and described in the legend to supplemental Fig. S4.