Enhanced ribonucleotide incorporation by an O-helix mutant of Thermus aquaticus DNA polymerase I

The O-helix of DNA polymerases has been implicated in substrate discriminat ion and replication fidelity. In this study, wild-type Thermus aquaticus DN A polymerase I (Taq pol I) and an O-helix mutant A661E was examined for the ir ability to discriminate between ribonucleotides and deoxyribonucleotides . Steady-state nucleotide extension kinetics were carried out using a templ ate cytidine and each nucleotide dNTP and rGTP. Wild-type Taq pol I and A66 1E demonstrated similar V-max and K-m values for the correct nucleotide dGT P. However, A661E discriminated between incorrect and correct nucleotide le ss well than wild-type: discrimination was reduced by factors of 9.5-, 5.6- and 15-fold for dATP, dTTP and rGTP, respectively. These data suggest that A661E is efficient polymerases in the presence of the correct deoxynucleot ide, dGTP, but it is impaired in ability to discriminate between correct an d incorrect deoxyribonucleotides or between ribo- and deoxyribonucleotides. A structural model of Taq pol I is described in which the mutation A661E a lters the interactions between the O-helix and the terminal two phosphate g roups in the primer strand. (C) 2001 Elsevier Science B.V. All rights reser ved.