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

Fingerprint

Taq Polymerase
DNA Polymerase I
Nucleotides
Thermus
Proline
Substitution reactions
Enzymes
Assays
Mutation
Kinetics
Experiments

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{5710aca15bc84d53a93f533232ce5ca1,
title = "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.",
author = "Aki Tosaka and Masanori Ogawa and Shonen Yoshida and Motoshi Suzuki",
year = "2001",
month = "7",
day = "20",
doi = "10.1074/jbc.M010635200",
language = "English",
volume = "276",
pages = "27562--27567",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "29",

}

O-helix mutant T664P of Thermus aquaticus DNA polymerase I : Altered catalytic properties for incorporation of incorrect nucleotides but not correct nucleotides. / Tosaka, Aki; Ogawa, Masanori; Yoshida, Shonen; Suzuki, Motoshi.

In: Journal of Biological Chemistry, Vol. 276, No. 29, 20.07.2001, p. 27562-27567.

Research output: Contribution to journalArticle

TY - JOUR

T1 - O-helix mutant T664P of Thermus aquaticus DNA polymerase I

T2 - Altered catalytic properties for incorporation of incorrect nucleotides but not correct nucleotides

AU - Tosaka, Aki

AU - Ogawa, Masanori

AU - Yoshida, Shonen

AU - Suzuki, Motoshi

PY - 2001/7/20

Y1 - 2001/7/20

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0035920224&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035920224&partnerID=8YFLogxK

U2 - 10.1074/jbc.M010635200

DO - 10.1074/jbc.M010635200

M3 - Article

C2 - 11346641

AN - SCOPUS:0035920224

VL - 276

SP - 27562

EP - 27567

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 29

ER -