Optimized Incorporation of an Unnatural Fluorescent Amino Acid Affords Measurement of Conformational Dynamics Governing High-fidelity DNA Replication

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2020 Oct 6

PMID 33020184

Citations 7

Authors

Tyler L Dangerfield

Kenneth A Johnson

Affiliations

Soon will be listed here.

Abstract

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 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.

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