Arg660Ser mutation in Thermus aquaticus DNA polymerase I suppresses T -> Ctransitions: implication of wobble base pair formation at the nucleotide incorporation step

We examined the replication fidelity of an Arg660Ser (R660S) mutant of Ther mus aquaticus DNA polymerase I (Taq pot I). In a forward mutation assay, R6 60S showed a marked reduction in T -->C transitions, one of the most freque nt errors made by the wild-type enzyme. Steady-state kinetics showed that R 660S discriminates against dGTP incorporation at a template T 13-fold bette r than the wild-type. R660S was also 3.2-fold less efficient than the wild- type at extending a T:dG mismatch. These results indicate that R660S has en hanced fidelity during incorporation and extension, which reduces its T --> C transition frequency. Interestingly, R660S also discriminated correct fro m incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs, 28-, 6.0-, 4.1- and 6.8-fold better, respectivel y, than the wild-type, although it may not always be as accurate as the wil d-type at the extension step. A structural model suggests that Arg660 may p articipate in two interactions that influence fidelity; the guanidinium gro up of Arg660 might interact with the incoming guanine base at the major gro ove and it might compete for forming another interaction with the primer te rminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data als o suggest that T:dGTP and C:dATP base pairs form 'wobble' structures at the incorporation step of Taq pot 1.