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

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.

• Authors: A Tosaka, M Ogawa, S Yoshida, M Suzuki
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 276 Issue 29 Pg. 27562-7 (Jul 20 2001) ISSN: 0021-9258 [Print] United States
• PMID: 11346641 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

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

Topics

• 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, genetics, metabolism)