Low fidelity mutants in the O-helix of Thermus aquaticus DNA polymerase I

Motoshi Suzuki, Amy K. Avicola, Leroy Hood, Lawrence A. Loeb

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

We screened 67 mutants in the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) for altered fidelity of DNA synthesis. These mutants were obtained (Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9670-9675) by substituting an oligonucleotide containing random sequences for codons 659-671, and selecting for complementation of a growth defect in Escherichia coli caused by temperature- sensitive host pol I. Thirteen mutants decreased fidelity in a screen that employed primer extension reactions lacking one of four complementary deoxynucleoside triphosphates (dNTPs). Three mutants were purified and exhibited 29-68% of wild-type specific activity. Homogeneous polymerases A661E, A661P, and T664R extended primers further than the wild-type, synthesizing past template nucleotides for which the complementary dNTP was absent. The data indicate that both misinsertion of incorrect nucleotides and extension of mispaired primer termini were increased. In a lacZα forward mutation assay, A661E and T664R yielded mutation frequencies at least 7- and 25-fold greater, respectively, than that of the wild-type polymerase. These findings emphasize the importance of the O-helix in substrate recognition and are compatible with a role for pyrophosphate release in enhancing fidelity of DNA synthesis.

Original languageEnglish
Pages (from-to)11228-11235
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number17
DOIs
Publication statusPublished - 25-04-1997
Externally publishedYes

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Taq Polymerase
DNA Polymerase I
Nucleotides
DNA
Mutation Rate
Codon
Oligonucleotides
Escherichia coli
Assays
Defects
Mutation
Temperature
Substrates
Growth
triphosphoric acid
diphosphoric acid

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Suzuki, Motoshi ; Avicola, Amy K. ; Hood, Leroy ; Loeb, Lawrence A. / Low fidelity mutants in the O-helix of Thermus aquaticus DNA polymerase I. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 17. pp. 11228-11235.
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Low fidelity mutants in the O-helix of Thermus aquaticus DNA polymerase I. / Suzuki, Motoshi; Avicola, Amy K.; Hood, Leroy; Loeb, Lawrence A.

In: Journal of Biological Chemistry, Vol. 272, No. 17, 25.04.1997, p. 11228-11235.

Research output: Contribution to journalArticle

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