Single-molecule Taq DNA polymerase dynamics

Abstract

Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics-regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template-deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-μs closures of Taq's fingers domain testing complementarity and orientation. On average, one transient closure was observed for every nucleotide binding event; even complementary substrate pairs averaged five transient closures between each catalytic incorporation at 72°C. The rate and duration of the transient closures and the catalytic events had almost no temperature dependence, leaving all of Taq's temperature sensitivity to its rate-determining open state.

Fig. 1.. A Taq-Intervening device schematic, atomic force microscopy (AFM) characterization, and sample signals generated at 72°C.

(A) The Taq-Intervening device featured an individual Taq DNA polymerase (green) site-specifically bioconjugated to a SWNT through a cysteine in the intervening domain (residue R411C, pink) to a pyrene-maleimide linker molecule (yellow). (B) AFM image of a sample device with a single Taq attachment (white arrow). Representative ΔI(t) signals generated in solutions of (C) activity buffer, and polyT with (D) mismatched (dTTP + dCTP + dGTP) or (E) matched (dATP) dNTPs. Two-level excursions only occurred when polyT and complementary dNTPs were both present. Protein Data Bank: 1TAQ (35).

Fig. 2.. Temperature dependence of ΔI(t) and individual catalytic closures.

Taq-Intervening with matched dATP and polyT generated different rates of catalytic closures at (A) 22°, (B) 45°, (C) 72°, and (D) 85°C. While the rate of catalytic closures increased with temperature (left), the duration of individual catalytic closures stayed constant (gray highlights and right). For clarity, individual data points represent 10-μs intervals; 1-μs resolution data are shown in fig. S7.

Fig. 3.. Taq-Intervening open and closed durations processing matched substrate (polyT + dATP).

(A) Probability distributions of the rate-determining waiting time, τ_open, with single-exponential fits. (B) Probability distributions of the catalytic closure durations, τ_cat, with double-exponential fits.

Fig. 4.. Taq-Fingers activity in matched and mismatched dNTPs at 45°C.

Taq-Fingers closures upon polyT with (A) matched dATP or (B) mismatched dGTP. Gray highlights the regions expanded at right with examples of individual catalytic closures (blue) and transient closures (red) at 1-μs resolution. Transient closures occurred in both matched dATP and mismatched dGTP, while catalytic closures only occurred with matched dATP. (C) Statistical distributions of closures per second in matched dATP (circles) and mismatched dGTP (diamonds) with 4-μs bin widths. The matched dATP data contained a combination of two types of events, whereas the mismatched dGTP only contained the transient closures. (Inset) On a longer time axis with 20-μs bins, the Taq-Fingers events denoted as catalytic closures overlapped the distribution of events measured with Taq-Intervening.

Fig. 5.. Variable kinetics of Taq-Fingers transient closures.

(A) Instantaneous rates calculated on 1-s intervals depict k_transient’s mean and range of variation with matched dATP (circles) or mismatched dGTP (diamonds). (B) Cumulative probability distributions on dual horizontal axes of rate (1/s, top) and relative energy (kcal/mol, bottom), overlaid with log-normal fits.

Fig. 6.. Distributions of event amplitudes <ΔI> generated by Taq-Fingers.

The amplitude <ΔI> of any event does not allow discrimination between matched (circles) and mismatched (diamonds) dNTPs nor between transient closures (red) and catalytic closures (blue). These four types of events had <ΔI> distributions with equal widths and peak positions. Here, event duration was used to separate events in matched dATP into either transient or catalytic closures. The top x axis provides a conversion of <ΔI> to its equivalent electrostatic gating ΔV_g, as determined from the device transconductance dI/dV_g. (Inset) Same distributions on a linear scale, showing the full width at half maximum of 9 mV.