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Originally published In Press as doi:10.1074/jbc.M011472200 on March 12, 2001

J. Biol. Chem., Vol. 276, Issue 22, 18836-18842, June 1, 2001
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The Conserved Active Site Motif A of Escherichia coli DNA Polymerase I Is Highly Mutable*

Akeo Shinkai, Premal H. Patel, and Lawrence A. LoebDagger

From the Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, Washington 98195-7705

Escherichia coli DNA polymerase I participates in DNA replication, DNA repair, and genetic recombination; it is the most extensively studied of all DNA polymerases. Motif A in the polymerase active site has a required role in catalysis and is highly conserved. To assess the tolerance of motif A for amino acid substitutions, we determined the mutability of the 13 constituent amino acids Val700-Arg712 by using random mutagenesis and genetic selection. We observed that every residue except the catalytically essential Asp705 can be mutated while allowing bacterial growth and preserving wild-type DNA polymerase activity. Hence, the primary structure of motif A is plastic. We present evidence that mutability of motif A has been conserved during evolution, supporting the premise that the tolerance for mutation is adaptive. In addition, our work allows identification of refinements in catalytic function that may contribute to preservation of the wild-type motif A sequence. As an example, we established that the naturally occurring Ile709 has a previously undocumented role in supporting sugar discrimination.

* This work was supported by National Institutes of Health Grants CA78885, CA39903, and CA74184 (to L. A. L.) and by a grant from Kyowa Hakko Kogyo Co., Ltd. (to A. S.).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.

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

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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