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

研究成果: Article査読

19 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)27562-27567
ページ数6
ジャーナルJournal of Biological Chemistry
276
29
DOI
出版ステータスPublished - 20-07-2001

All Science Journal Classification (ASJC) codes

  • 生化学
  • 分子生物学
  • 細胞生物学

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