Using 2-Aminopurine Fluorescence to Measure Incorporation of Incorrect Nucleotides by Wild Type and Mutant Bacteriophage T4 DNA Polymerases

• Published in: The Journal of biological chemistry 2002-10, Vol.277 (43), p.40640-40649
• Main Authors: Fidalgo da Silva, Elizabeth, Mandal, Subhrangsu S., Reha-Krantz, Linda J.
• Format: Article
• Language: English
• Subjects: 2-Aminopurine - chemistry; Bacteriophage T4 - enzymology; Base Sequence; DNA Primers; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; Fluorescence; Kinetics; Mutation; Thymine Nucleotides - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M203315200

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 (∼367 μ m ) than for incorporation of dTMP opposite template A (∼31 μ 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 an error avoidance mechanism to prevent incorporation of incorrect nucleotides.