FIGURE 4.

Asp⁷⁰⁵ and Asp⁸⁸² are not required for the rapid template strand conformational transition detected by 2-AP. The plots show the stopped-flow fluorescence signal upon the addition of dGTP, at the indicated concentrations, to the 2-AP-containing DNA duplex L:T(+1)2-AP:3′H (Fig. 3B) in complex with WT (A), D705A (B), and D882A Pol I(KF) (C). In the absence of protein, the addition of dGTP (1, 10, and 100 μm) to the 2-AP-DNA gave no fluorescence change (data not shown). Only the first 0.1 s of data are plotted in order to show rapid changes. The black lines superimposed on the data traces show fitting to a double exponential, taking account of the fluorescence changes that occur in the instrument dead time (see supplemental Fig. S3 and Table S2).