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

M. Ogawa, A. Tosaka, Y. Ito, S. Yoshida, Motoshi Suzuki

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)197-207
Number of pages11
JournalMutation Research - DNA Repair
Volume485
Issue number3
DOIs
Publication statusPublished - 04-04-2001
Externally publishedYes

Fingerprint

Taq Polymerase
Ribonucleotides
DNA Polymerase I
Deoxyribonucleotides
Nucleotides
Cytidine
Structural Models
DNA-Directed DNA Polymerase
Phosphates
Mutation
Kinetics
Substrates
deoxyguanosine triphosphate

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Toxicology
  • Genetics

Cite this

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abstract = "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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Enhanced ribonucleotide incorporation by an O-helix mutant of Thermus aquaticus DNA polymerase I. / Ogawa, M.; Tosaka, A.; Ito, Y.; Yoshida, S.; Suzuki, Motoshi.

In: Mutation Research - DNA Repair, Vol. 485, No. 3, 04.04.2001, p. 197-207.

Research output: Contribution to journalArticle

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