FIGURE 5.

Asp⁷⁰⁵ is not required for fingers-closing activity. Stopped-flow fluorescence used 744-AEDANS Pol I(KF) and the dabcyl-containing DNA duplex, H:T(−8)D:3′H (Fig. 3C) in Mg²⁺ or Mn²⁺ buffer. The binary complex of labeled WT Pol I(KF) and DNA in 5 mm Mg²⁺(A) or 2 mm Mn²⁺ buffer (C) was mixed with dTTP to give the indicated final concentrations. B and D, analogous plots obtained with D705A Pol I(KF) in 10 mm Mg²⁺ or 10 mm Mn²⁺ buffer, respectively. Time is plotted on a logarithmic scale in order to illustrate clearly all phases of the reaction. The black lines superimposed on the data traces show fitting to a double exponential for the wild-type reactions or to a single exponential for the D705A reactions (see supplemental Fig. S5 and Table S3 for curve fitting and residuals), yielding the reaction rates and amplitudes reported in Table 3. In the absence of the dabcyl quencher on DNA, essentially no change in AEDANS fluorescence was observed (supplemental Fig. S6A).