Figure 4.

Presteady state incorporation of (R/S)-β,γ-CHCl-dGTP into single-gapped DNA. (A) Correct incorporation opposite dC of R (left) and S (right) CHCl diastereomers. The cartoon shows the generalized scheme for the reaction. A plot of the percentage of primer extension vs. time for six concentrations of the CHCl analogue is shown for one of three repeats of each reaction (1 μM, filled circles; 2 μM, open circles; 4 μM, filled triangles; 8 μM, open triangles; 16 μM, filled squares; 32 μM, open squares). Below each plot is a time course shown on a representative gel for the first concentration (1 μM), where P represents the unextended primer and P+1 the extension of the primer by a single incorporation. The corresponding reaction time is shown below each lane. Below these gels is a plot of the observed rate constant vs. the corresponding analogue concentration for both diastereomers. (B) Misincorporation opposite dT of R (left) and S (right) CHCl diastereomers. As for the correct pairing, the percentage of primer extension vs. time for each concentration of analogue is shown for both diastereomers (25 μM, filled circles; 50 μM, open circles; 100 μM, filled triangles; 200 μM, open triangles; 400 μM, filled squares; 600 μM, open squares) with a representative gel for each set of reactions; the corresponding reaction time is shown below each lane. The observed rate constant is plotted vs. the corresponding analogue concentration on the same plot for each diastereomer below. The reactions were carried out in triplicate and have an SEM of ± 15%; the figure shows a representative data set.