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Mutants affecting nucleotide recognition by T7 DNA polymerase

M J Donlin1, K A Johnson

  • 1Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802.

Biochemistry
|December 13, 1994
PubMed
Summary

Mutations in bacteriophage T7 DNA polymerase active site residues Glu480 and Tyr530 affect nucleotide binding, particularly for dTTP. These changes slightly reduce enzyme fidelity, impacting DNA replication accuracy.

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Biochemistry

Background:

  • Bacteriophage T7 DNA polymerase is a key enzyme for DNA replication.
  • Nucleotide selection is a critical step in DNA synthesis, directly impacting fidelity.
  • Conserved residues in the polymerase active site play crucial roles in substrate recognition and binding.

Purpose of the Study:

  • To investigate the roles of conserved residues Glu480 and Tyr530 in the active site of T7 DNA polymerase (exo-) in nucleotide selection.
  • To characterize the kinetic and equilibrium properties of E480D(exo-) and Y530F(exo-) mutants.
  • To determine the impact of these mutations on DNA binding, nucleotide incorporation, pyrophosphorolysis, and fidelity.

Main Methods:

  • Site-directed mutagenesis was used to create Glu480-Asp and Tyr530-Phe mutations in an exonuclease-deficient T7 DNA polymerase.

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  • Single-turnover experiments employing rapid chemical quench-flow methods were used to measure kinetic and equilibrium constants.
  • Analysis included DNA binding, nucleotide incorporation, pyrophosphorolysis, and misincorporation assays.

    • Main Results:

    • Both E480D(exo-) and Y530F(exo-) mutants exhibited slightly reduced DNA binding affinity (lower Kd values).
    • Mutations significantly decreased dTTP binding affinity (40-fold for E480D, 8-10-fold for Y530F), with E480 showing a specific destabilization of dTTP binding.
    • Misincorporation assays revealed saturation kinetics with incorrect nucleotides for both mutants, indicating slightly lower fidelity compared to wild-type.

    Conclusions:

    • Residues Glu480 and Tyr530 are important for ground-state nucleotide binding in T7 DNA polymerase.
    • Glu480 appears to specifically stabilize incoming dTTP during A:T base pair formation, potentially compensating for fewer hydrogen bonds.
    • The mutations lead to a modest decrease in overall polymerase fidelity.