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J. Biol. Chem., Vol. 281, Issue 4, 2289-2295, January 27, 2006

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Functional Evidence for a Small and Rigid Active Site in a High Fidelity DNA Polymerase

PROBING T7 DNA POLYMERASE WITH VARIABLY SIZED BASE PAIRS^*

Tae Woo Kim1, Luis G. Brieba2, Tom Ellenberger, and Eric T. Kool3

From the Department of Chemistry, Stanford University, Stanford, California 94305 and the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Hypotheses on the origins of high fidelity in replicative DNA polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 Å) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1:1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 Å beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 Å. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative polymerases and that a less rigid (looser) and larger active site can lead to lower fidelity.

Received for publication, October 3, 2005 , and in revised form, November 22, 2005.

^* This work was supported in part by National Institutes of Health Grants GM072705 (to E. T. K.) and GM55390 (to T. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Figs. S1-S3.

¹ Recipient of a Korea Science and Engineering Foundation fellowship.

² Recipient of a Pew Latinoamerican Scholar fellowship.

³ To whom correspondence should be addressed: Dept. of Chemistry, Stanford University, Stanford, CA 94305-5080. Tel.: 650-724-4741; Fax: 650-725-0259; E-mail: kool{at}stanford.edu.

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