Conformational dynamics during high-fidelity DNA replication and translocation defined using a DNA polymerase with a fluorescent artificial amino acid

Abstract

We address the role of enzyme conformational dynamics in specificity for a high-fidelity DNA polymerase responsible for genome replication. We present the complete characterization of the conformational dynamics during the correct nucleotide incorporation forward and reverse reactions using stopped-flow and rapid-quench methods with a T7 DNA polymerase variant containing a fluorescent unnatural amino acid, (7-hydroxy-4-coumarin-yl) ethylglycine, which provides a signal for enzyme conformational changes. We show that the forward conformational change (>6000 s −1 ) is much faster than chemistry (300 s −1 ) while the enzyme opening to allow release of bound nucleotide (1.7 s −1 ) is much slower than chemistry. These parameters show that the conformational change selects a correct nucleotide for incorporation through an induced-fit mechanism. We also measured conformational changes occurring after chemistry and during pyrophosphorolysis, providing new insights into processive polymerization. Pyrophosphorolysis occurs via a conformational selection mechanism as the pyrophosphate binds to a rare pretranslocation state of the enzyme–DNA complex. Global data fitting was achieved by including experiments in the forward and reverse directions to correlate conformational changes with chemical reaction steps. This analysis provided well-constrained values for nine rate constants to establish a complete free-energy profile including the rates of DNA translocation during processive synthesis. Translocation does not follow Brownian ratchet or power stroke models invoking nucleotide binding as the driving force. Rather, translocation is rapid and thermodynamically favorable after enzyme opening and pyrophosphate release, and it appears to limit the rate of processive synthesis at 4 °C.

Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid

“…This study completes our prior characterization of the role of enzyme conformational dynamics in DNA polymerase nucleotide specificity (12,13,31,42) by extending our analysis of a high fidelity DNA polymerase to include the kinetics of misincorporation. Prior analysis of nucleotide-induced conformational changes using the T7 DNA polymerase E514Cou variant showed that for a correct base pair, the transition from the open to closed enzyme state was at least 20-fold faster than chemistry, but the reverse conformational change to allow release of the nucleotide was much slower than chemistry.…”

“…While k cat / K m values for a correct- and a mismatched-nucleotide incorporation have been reported (9,29–31,33,34), general questions about misincorporation still remain, especially for high fidelity DNA polymerases. Are certain mismatches incorporated more efficiently than others?…”

“…To fully understand the role of induced-fit in DNA polymerase fidelity, we need to compare the kinetics of conformational changes during misincorporation with those during correct incorporation (31). In our previous work, we found that for correct nucleotide incorporation, all nucleotides gave a fluorescence signal that reflected changes in enzyme structure before and after incorporation (30).…”

“…We also derived expressions for the parameters k cat , K m , and k cat / K m for each reaction scheme using the King-Altman method (44) and they are listed to the right of each free energy profile in Figure 8. For comparison we also present the free energy profile for correct nucleotide incorporation in Figure 8A from rate constants determined in our previous study on the T7 DNA polymerase E514Cou variant (31), without the translocation and competitive PPi binding for simplicity. Although we recognize the translocation step must be occurring in our model for misincorporation, the experimental data do not define this step based on the experiments presented in this paper and it is expected to be much faster than the misincorporation reaction.…”

“…A) Free energy profile for correct nucleotide incorporation. Rate constants used to make this free energy profile are from (31). The highest barrier relative to the starting material is for the conformational change step (EDA to FDA states).…”