Site-specific labeling of T7 DNA polymerase with a conformationally sensitive fluorophore and its use in detecting single-nucleotide polymorphisms.
Like most enzymes, DNA polymerases undergo a large conformational change on the binding of a correct nucleotide. To determine how the conformational change contributes to substrate specificity, we labeled the T7 DNA polymerase with a conformationally sensitive fluorophore at a position that provides a signal coincident with structural changes following nucleotide binding and distinguishes correct base pairs from incorrect ones by the sign of the fluorescence change. Here we describe methods to document that only one site on the polymerase was labeled with the fluorophore based on mass spectral analysis of tryptic peptides. In addition, we show by equilibrium titrations of opposing signals that mismatches and correct bases compete for the same site. This analysis forms an essential basis for characterization of a fluorescently labeled enzyme intended for mechanistic studies. Finally, we show that the labeled enzyme can be used to identify single-nucleotide mutations in a procedure that could be automated.
Tsai
Yu-Chih
YC
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.
Jin
Zhinan
Z
Johnson
Kenneth A
KA
D017123:Bacteriophage T7 / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D011487:Protein Conformation; D013194:Staining and Labeling / Q000379:methods
D003374:Coumarins; D005456:Fluorescent Dyes; C088410:N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase