O-helix mutant T664P of Thermus aquaticus DNA polymerase I: altered catalytic properties for incorporation of incorrect nucleotides but not correct nucleotides

J Biol Chem. 2001 Jul 20;276(29):27562-7. doi: 10.1074/jbc.M010635200. Epub 2001 May 9.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Catalysis
  • Catalytic Domain
  • Crystallography, X-Ray
  • DNA Primers
  • DNA, Recombinant
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Nucleotides / metabolism*
  • Protein Conformation
  • Taq Polymerase / chemistry
  • Taq Polymerase / genetics
  • Taq Polymerase / metabolism*


  • DNA Primers
  • DNA, Recombinant
  • Nucleotides
  • Taq Polymerase