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.
Highlights
DNA polymerases are ideal systems to study enzyme specificity because fidelity is physiologically important and alternative substrates are well known
When the conformational change is faster than the chemistry, the question as to whether the structural change directly influences specificity must be based on complete kinetic analysis of the pathway
Previous attempts to examine the kinetics of the conformational change using T7 DNA polymerase were flawed because they were based on constructing a cys-lite mutant to afford site-specific labeling with a fluorescent probe [1]
Summary
We first measured the kinetics and equilibrium for nucleotide binding steps (K1, k2, and k−2 in Equation 2) using a 2’,3’ dideoxy-nucleotide-terminated primer to allow dATP binding but prevent the chemical reaction. The nucleotide binding rate was measured by mixing a preformed labeled E-DNAdd complex with various concentrations of dATP (5–100 μM) and Mg2+ in the stopped flow (Fig. 2A). To more accurately measure the rate constant for nucleotide dissociation (limited by enzyme opening, k−2), a stopped flow experiment was performed where a solution containing a ternary E.DNAdd.dATP complex was mixed with a large excess of unlabeled wildtype E.DNA complex to start the reaction. The small initial fast phase is due to the small amount of unbound nucleotide from the E.DNAdd.dATP mixture that is rapidly incorporated by the wildtype polymerase upon mixing, while the slower phase reflects the nucleotide dissociation rate, which is limited by the reverse of the conformational change step (k−2) from the labeled enzyme. The net Kd,net determined from the rate constants for the fit to the on- and off-rate experiments for a two-step binding model is defined by Equation 5
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