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.
authors.affiliation: Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, 466-8550, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Tosaka
authors.forename: M
authors.initials: M
authors.lastname: Ogawa
authors.forename: S
authors.initials: S
authors.lastname: Yoshida
authors.forename: M
authors.initials: M
authors.lastname: Suzuki
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004274:DNA, Recombinant; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D019914:Taq Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004274:DNA, Recombinant; D009711:Nucleotides; D019914:Taq Polymerase