Abstract
Previous studies indicate that the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) plays an important role in the replication fidelity of the enzyme. This study examines the role of Thr-664, which lies in the middle of the O-helix of Taq pol I. A mutant of Taq Pol I with a proline substitution of Thr-664 (T664P) exhibits much lower replication fidelity than the wild type enzyme in a forward mutation assay. T664P produces base substitution, single-base deletion, and single-base insertion errors at 20-, 5, and 50-fold higher rates than wild type, respectively. In specific activity and steady-state kinetic experiments, T664P was catalytically robust for insertion of correct nucleotides. In contrast, it incorporated incorrect nucleotides 6.1- to 10-fold more efficiently than wild type at a template dC. Mismatched primer termini were extended by T664P 4.2- to 9.5-fold more efficiently than wild type. These data imply that the O-helix with a proline at position 664 functions like wild type Taq pol I for correct nucleotide incorporations, but bends and enlarges the catalytic pocket of the enzyme and increases the rate of nucleotide misincorporation.
Highlights
Previous studies indicate that the O-helix of Thermus aquaticus (Taq) DNA polymerase I plays an important role in the replication fidelity of the enzyme
This study examines the role of Thr-664, which lies in the middle of the O-helix of Taq polymerase I (pol I)
In the crystal structure models of Thermus aquaticus (Taq) DNA polymerase I that are demonstrated by Li et al [14], we found that substitution of Thr-664 with proline (T664P) removes at least two hydrogen bonds in the closed complex structure (Fig. 1)
Summary
Materials—–Wild type and mutants in Taq pol I were purified as described previously [7]. The magnesium concentration was raised to 7 mM to force misincorporation [10] These changes in assay conditions made it possible to measure the mutation frequency of wild type polymerases and provided a sufficient number of mutant plaques for analysis. In reactions for correct nucleotide incorporation, concentrations of wild-type protein and substrate were 0.3 nM and 0.1–5 M dGTP, 2 nM and 0.5–20 M dATP, 2 nM and 0.5–20 M dTTP, and 0.5 nM and 0.5–20 M dCTP, respectively. In reactions for incorrect nucleotide incorporation at template C, concentrations of wild-type protein and substrate were 120 nM and 0.2–50 M dATP, 120 nM and 20 –200 M dTTP, and 230 nM and 100 –500 M dCTP. The polymerase-DNA equilibrium binding constants (KD) were determined by the kinetic values using two concentrations (50 and 5 nM) of template-primer [13]
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