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Originally published In Press as doi:10.1074/jbc.M203315200 on August 19, 2002

J. Biol. Chem., Vol. 277, Issue 43, 40640-40649, October 25, 2002
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Using 2-Aminopurine Fluorescence to Measure Incorporation of Incorrect Nucleotides by Wild Type and Mutant Bacteriophage T4 DNA Polymerases*

Elizabeth Fidalgo da SilvaDagger , Subhrangsu S. Mandal§, and Linda J. Reha-Krantz

From the Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada

The ability of wild type and mutant T4 DNA polymerases to discriminate in the utilization of the base analog 2-aminopurine (2AP) and the fluorescence of 2AP were used to determine how DNA polymerases distinguish between correct and incorrect nucleotides. Because T4 DNA polymerase incorporates dTMP opposite 2AP under single-turnover conditions, it was possible to compare directly the kinetic parameters for incorporation of dTMP opposite template 2AP to the parameters for incorporation of dTMP opposite template A without the complication of enzyme dissociation. The most significant difference detected was in the Kd for dTTP, which was 10-fold higher for incorporation of dTMP opposite template 2AP (~367 µM) than for incorporation of dTMP opposite template A (~31 µM). In contrast, the dTMP incorporation rate was reduced only about 2-fold from about 318 s-1 with template A to about 165 s-1 for template 2AP. Discrimination is due to the high selectivity in the initial nucleotide-binding step. T4 DNA polymerase binding to DNA with 2AP in the template position induces formation of a nucleotide binding pocket that is preshaped to bind dTTP and to exclude other nucleotides. If nucleotide binding is hindered, initiation of the proofreading pathway acts as an error avoidance mechanism to prevent incorporation of incorrect nucleotides.

* This work was supported in part by grants from the Canadian Institutes of Health Research Grant MOP-14300 and the Alberta Heritage Foundation for Medical Research (to L. J. R.-K.).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 Supported CAPES (Brasília-Brazil) and the Universidade Gama Filho, Rio de Janeiro, Brazil.

§ Supported by an Alberta Heritage Foundation for Medical Research postdoctoral fellowship.

Scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed. Tel.: 780-492-5383; Fax: 780-492-2216; E-mail: LREHA@gpu.srv.ualberta.ca.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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