Using 2-aminopurine fluorescence to measure incorporation of incorrect nucleotides by wild type and mutant bacteriophage T4 DNA polymerases.

Abstract:

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 K(d) for dTTP, which was 10-fold higher for incorporation of dTMP opposite template 2AP (approximately 367 microm) than for incorporation of dTMP opposite template A (approximately 31 microm). 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.

Polymerases:

Topics:

Mutational Analysis, Biotech Applications, Kinetic Parameters, Nucleotide Analogs / Template Lesions, Fidelity, Nucleotide Incorporation, Methods

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 L412M Fidalgo da Silva E2002 Nucleotide Substitution Rate 7.7 Mutation frequency (relative to WT) Technique: rII+ reversion (2AP (1 mg/ml))
T4 I417V Fidalgo da Silva E2002 Nucleotide Substitution Rate 0.1 Mutation frequency (relative to WT) Technique: rII+ reversion (2AP (1 mg/ml))
T4 D112AE114A Fidalgo da Silva E2002 Kd 31uM Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Rapid quench (opposite template A)

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