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Papers In Press, published online ahead of print August 19, 2002
J. Biol. Chem, 10.1074/jbc.M203315200
Submitted on April 6, 2002
Revised on July 29, 2002
Accepted on August 19, 2002
Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9
Corresponding Author: LREHA{at}gpu.srv.ualberta.ca
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. Since 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 
M) than for incorporation of dTMP opposite template A (31 M). 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 error avoidance mechanism to prevent incorporation of incorrect nucleotides.
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