Low Fidelity Mutants in the O-Helix of Thermus aquaticus DNA Polymerase I

• Published in: The Journal of biological chemistry 1997-04, Vol.272 (17), p.11228-11235
• Main Authors: Suzuki, M, Avicola, A K, Hood, L, Loeb, L A
• Format: Article
• Language: English
• Subjects: Base Sequence; DNA Polymerase I - genetics; DNA Polymerase I - metabolism; DNA Replication; DNA, Bacterial - biosynthesis; Escherichia coli; Models, Molecular; Molecular Sequence Data; Mutation; Protein Structure, Secondary; Selection, Genetic; Structure-Activity Relationship; Thermus - enzymology; Thermus - genetics; Thermus aquaticus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.17.11228

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.