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Originally published In Press as doi:10.1074/jbc.M000097200 on July 20, 2000

J. Biol. Chem., Vol. 275, Issue 42, 32728-32735, October 20, 2000
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Thermus aquaticus DNA Polymerase I Mutants with Altered Fidelity
INTERACTING MUTATIONS IN THE O-HELIX*

Motoshi SuzukiDagger §, Shonen YoshidaDagger , Elinor T. Adman, A. Blank§, and Lawrence A. Loeb§||

From Dagger  Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, 466-8550, Japan, the § Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, Washington 98195-7705, and the  Department of Biological Structure, University of Washington, Seattle, Washington 98195-7705

Phe667 in the conserved O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) is known to be important for discrimination against dideoxy-NTPs. We show here that Phe667 is also important for base selection fidelity. In a forward mutation assay at high polymerase concentration, wild type pol I catalyzed frequent A right-arrow T and G right-arrow T transversions and -1 frameshifts at nonreiterated sites involving loss of a purine immediately downstream of a pyrimidine. The mutants F667L and A661E,I665T,F667L exhibited large decreases in A right-arrow T and G right-arrow T transversions, and the triple mutant displayed reduction in the aforementioned -1 frameshifts as well. Kinetic analysis showed that the F667L and A661E,I665T,F667L polymerases discriminated against synthesis of A:A mispairs more effectively and catalyzed less extension of A:A mispairs than the wild type enzyme. These data indicate that Phe667 functions in maintaining the error frequency and spectrum, and the catalytic efficiency, of wild type pol I. We also found that the strong general mutator activity conferred by the single A661E substitution was entirely suppressed in the A661E, I665T,F667L polymerase, exemplifying how interactions among O-helix residues can contribute to fidelity. We discuss the mutator and anti-mutator mutations in light of recently obtained three-dimensional structures of T. aquaticus pol I.

* This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (to S. Y. and M. S.), by University of Washington Center Grant P30 ES07033 from the National Institute of Environmental Health Sciences (to E. T. A.), and by Grants CA78885, AG01751, and CA74184 from the National Institutes of Health (to L. A. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Joseph Gottstein Memorial Cancer Research Lab., Dept. of Pathology, Box 357705, University of Washington, Seattle, WA 98195-7705. Tel.: 206-543-6015; Fax: 206-543-3967; E-mail: laloeb@u.washington.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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