FIGURE 6.

D882A inhibits fingers-closing, and dNTPs promote DNA dissociation from the open complex. A, stopped-flow fluorescence used 744-AEDANS D882A Pol I(KF) and the H:T(−8)D:3′H DNA duplex (Fig. 3C). The binary complex of labeled protein and DNA was mixed with the complementary nucleotide, dTTP, to give the indicated final concentrations. The buffer in both syringes included 10 mm MgCl₂. B, the rate of dissociation of 744-AEDANS D882A Pol I(KF) from H:T(−8)D:3′OH DNA (Fig. 3C) was measured using a DNA trap as described under “Experimental Procedures.” The red trace is the dissociation of the Pol-DNA binary complex when mixed with an excess of an unmodified DNA duplex (DNA trap). The blue trace shows the dissociation of the correctly paired ternary complex, measured by including a 2 mm concentration of the complementary nucleotide, dTTP, in the DNA trap solution. The green trace shows the dissociation of a mismatched ternary complex, when the DNA trap solution contained a 2 mm concentration of a noncomplementary nucleotide, dGTP. C, as in A, except that the reaction buffer contained 10 mm MnCl₂ instead of MgCl₂. The black lines superimposed on the data traces in A and B show fitting of the fluorescence increases to single exponential equations, with rates and amplitudes reported in supplemental Table S3.