Thermus aquaticus DNA polymerase I mutants with altered fidelity. Interacting mutations in the O-helix

Motoshi Suzuki, S. Yoshida, E. T. Adman, A. Blank, L. A. Loeb, J. Gottstein

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

Phe667 in the conserved O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) is known to be important for discrimination against dideoxy-NTPs. We show here that Phe667 is also important for base selection fidelity. In a forward mutation assay at high polymerase concentration, wild type pol I catalyzed frequent A → T and G → T transversions and -1 frameshifts at nonreiterated sites involving loss of a purine immediately downstream of a pyrimidine. The mutants F667L and A661E,I665T, F667L exhibited large decreases in A → T and G → T transversions, and the triple mutant displayed reduction in the aforementioned -1 frameshifts as well. Kinetic analysis showed that the F667L and A661E,I665T, F667L polymerases discriminated against synthesis of A:A mispairs more effectively and catalyzed less extension of A:A mispairs than the wild type enzyme. These data indicate that Phe667 functions in maintaining the error frequency and spectrum, and the catalytic efficiency, of wild type pol I. We also found that the strong general mutator activity conferred by the single A661E substitution was entirely suppressed in the A661E, I665T,F667L polymerase, exemplifying how interactions among O-helix residues can contribute to fidelity. We discuss the mutator and anti-mutator mutations in light of recently obtained three-dimensional structures of T. aquaticus pol I.

Original languageEnglish
Pages (from-to)32728-32735
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number42
DOIs
Publication statusPublished - 20-10-2000
Externally publishedYes

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Taq Polymerase
DNA Polymerase I
Assays
Substitution reactions
Mutation
Kinetics
Enzymes
purine
pyrimidine

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Suzuki, Motoshi ; Yoshida, S. ; Adman, E. T. ; Blank, A. ; Loeb, L. A. ; Gottstein, J. / Thermus aquaticus DNA polymerase I mutants with altered fidelity. Interacting mutations in the O-helix. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 42. pp. 32728-32735.
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Thermus aquaticus DNA polymerase I mutants with altered fidelity. Interacting mutations in the O-helix. / Suzuki, Motoshi; Yoshida, S.; Adman, E. T.; Blank, A.; Loeb, L. A.; Gottstein, J.

In: Journal of Biological Chemistry, Vol. 275, No. 42, 20.10.2000, p. 32728-32735.

Research output: Contribution to journalArticle

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T1 - Thermus aquaticus DNA polymerase I mutants with altered fidelity. Interacting mutations in the O-helix

AU - Suzuki, Motoshi

AU - Yoshida, S.

AU - Adman, E. T.

AU - Blank, A.

AU - Loeb, L. A.

AU - Gottstein, J.

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N2 - Phe667 in the conserved O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) is known to be important for discrimination against dideoxy-NTPs. We show here that Phe667 is also important for base selection fidelity. In a forward mutation assay at high polymerase concentration, wild type pol I catalyzed frequent A → T and G → T transversions and -1 frameshifts at nonreiterated sites involving loss of a purine immediately downstream of a pyrimidine. The mutants F667L and A661E,I665T, F667L exhibited large decreases in A → T and G → T transversions, and the triple mutant displayed reduction in the aforementioned -1 frameshifts as well. Kinetic analysis showed that the F667L and A661E,I665T, F667L polymerases discriminated against synthesis of A:A mispairs more effectively and catalyzed less extension of A:A mispairs than the wild type enzyme. These data indicate that Phe667 functions in maintaining the error frequency and spectrum, and the catalytic efficiency, of wild type pol I. We also found that the strong general mutator activity conferred by the single A661E substitution was entirely suppressed in the A661E, I665T,F667L polymerase, exemplifying how interactions among O-helix residues can contribute to fidelity. We discuss the mutator and anti-mutator mutations in light of recently obtained three-dimensional structures of T. aquaticus pol I.

AB - Phe667 in the conserved O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) is known to be important for discrimination against dideoxy-NTPs. We show here that Phe667 is also important for base selection fidelity. In a forward mutation assay at high polymerase concentration, wild type pol I catalyzed frequent A → T and G → T transversions and -1 frameshifts at nonreiterated sites involving loss of a purine immediately downstream of a pyrimidine. The mutants F667L and A661E,I665T, F667L exhibited large decreases in A → T and G → T transversions, and the triple mutant displayed reduction in the aforementioned -1 frameshifts as well. Kinetic analysis showed that the F667L and A661E,I665T, F667L polymerases discriminated against synthesis of A:A mispairs more effectively and catalyzed less extension of A:A mispairs than the wild type enzyme. These data indicate that Phe667 functions in maintaining the error frequency and spectrum, and the catalytic efficiency, of wild type pol I. We also found that the strong general mutator activity conferred by the single A661E substitution was entirely suppressed in the A661E, I665T,F667L polymerase, exemplifying how interactions among O-helix residues can contribute to fidelity. We discuss the mutator and anti-mutator mutations in light of recently obtained three-dimensional structures of T. aquaticus pol I.

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