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

Authors
Type
Published Article
Journal
The Journal of biological chemistry
Publication Date
Volume
275
Issue
42
Pages
32728–32735
Identifiers
PMID: 10906120
Source
Medline

Abstract

Phe(667) 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 Phe(667) 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 Phe(667) 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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