Optimized incorporation of an unnatural fluorescent amino acid affords measurement of conformational dynamics governing high-fidelity DNA replication
| Autor: | Tyler L. Dangerfield, Kenneth A. Johnson |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
DNA Replication
0301 basic medicine Conformational change Fluorophore DNA polymerase Glycine DNA-Directed DNA Polymerase Biochemistry 03 medical and health sciences chemistry.chemical_compound Coumarins Escherichia coli Nucleotide Enzyme kinetics Molecular Biology Fluorescent Dyes chemistry.chemical_classification 030102 biochemistry & molecular biology biology Protein dynamics DNA replication DNA Cell Biology Amino acid 030104 developmental biology chemistry Enzymology biology.protein Biophysics |
| Zdroj: | J Biol Chem |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.ra120.015557 |
| Popis: | DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli. MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication. |
| Databáze: | OpenAIRE |
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