TY - JOUR
T1 - Enhanced ribonucleotide incorporation by an O-helix mutant of Thermus aquaticus DNA polymerase I
AU - Ogawa, M.
AU - Tosaka, A.
AU - Ito, Y.
AU - Yoshida, S.
AU - Suzuki, M.
N1 - Funding Information:
We thank Hong-Yan Zhu for their initial involvement in the work. We are grateful to A. Blank and E. Adman at University of Washington for helpful discussion. This study was supported by Grants-in Aid from the Ministry of Education, Science, Sports and Culture of Japan to SY and MS.
PY - 2001/4/4
Y1 - 2001/4/4
N2 - The O-helix of DNA polymerases has been implicated in substrate discrimination and replication fidelity. In this study, wild-type Thermus aquaticus DNA polymerase I (Taq pol I) and an O-helix mutant A661E was examined for their ability to discriminate between ribonucleotides and deoxyribonucleotides. Steady-state nucleotide extension kinetics were carried out using a template cytidine and each nucleotide dNTP and rGTP. Wild-type Taq pol I and A661E demonstrated similar Vmax and Km values for the correct nucleotide dGTP. However, A661E discriminated between incorrect and correct nucleotide less 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 deoxynucleotide, dGTP, but it is impaired in ability to discriminate between correct and incorrect deoxyribonucleotides or between ribo- and deoxyribonucleotides. A structural model of Taq pol I is described in which the mutation A661E alters the interactions between the O-helix and the terminal two phosphate groups in the primer strand.
AB - The O-helix of DNA polymerases has been implicated in substrate discrimination and replication fidelity. In this study, wild-type Thermus aquaticus DNA polymerase I (Taq pol I) and an O-helix mutant A661E was examined for their ability to discriminate between ribonucleotides and deoxyribonucleotides. Steady-state nucleotide extension kinetics were carried out using a template cytidine and each nucleotide dNTP and rGTP. Wild-type Taq pol I and A661E demonstrated similar Vmax and Km values for the correct nucleotide dGTP. However, A661E discriminated between incorrect and correct nucleotide less 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 deoxynucleotide, dGTP, but it is impaired in ability to discriminate between correct and incorrect deoxyribonucleotides or between ribo- and deoxyribonucleotides. A structural model of Taq pol I is described in which the mutation A661E alters the interactions between the O-helix and the terminal two phosphate groups in the primer strand.
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U2 - 10.1016/S0921-8777(00)00081-1
DO - 10.1016/S0921-8777(00)00081-1
M3 - Article
C2 - 11267831
AN - SCOPUS:0035804793
SN - 0921-8777
VL - 485
SP - 197
EP - 207
JO - Mutation Research - DNA Repair
JF - Mutation Research - DNA Repair
IS - 3
ER -