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

Aki Tosaka, Masanori Ogawa, Shonen Yoshida, Motoshi Suzuki

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)27562-27567
Number of pages6
JournalJournal of Biological Chemistry
Volume276
Issue number29
DOIs
Publication statusPublished - 20-07-2001
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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