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

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 po l I catalyzed frequent A --> T and G --> T transversions and -1 frameshifts at nonreiterated sites involving loss of a purine immediately downstream o f a pyrimidine, The mutants F667L and A661E,I665T, F667L exhibited large de creases in A --> T and G --> T transversions, and the triple mutant display ed reduction in the aforementioned -1 frameshifts as well. Kinetic analysis showed that the F667L and A661E,I665T, F667L polymerases discriminated aga inst synthesis of A:A mispairs more effectively and catalyzed less extensio n of A:A mispairs than the wild type enzyme. These data indicate that Phe(6 67) functions in maintaining the error frequency and spectrum, and the cata lytic efficiency, of wild type pol I. We also found that the strong general mutator activity conferred by the single A661E substitution was entirely s uppressed in the A661E, I665T,F667L polymerase, exemplifying how interactio ns among O-helix residues can contribute to fidelity. We discuss the mutato r and anti-mutator mutations in light of recently obtained three-dimensiona l structures of T, aquaticus pol I.