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 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.
Highlights
The role of substrate-induced conformational changes in enzyme specificity has been a controversial topic for decades [1]
We show that the fluorescence is sensitive to conformational changes of the protein during nucleotide incorporation (Fig. 1)
To circumvent the problems associated with the cysteine labeling strategy used for the 8-Cys-lite variant [18], we tested the amber suppression to insert a site-specific unnatural amino acid, requiring only one residue substitution in the protein to directly incorporate a fluorescent unnatural amino acid into the fingers domain of the T7 DNA polymerase (Fig. 1)
Summary
The role of substrate-induced conformational changes in enzyme specificity has been a controversial topic for decades [1]. The original T7 DNA polymerase papers were published almost 30 years ago, measuring the correct incorporation [2], misincorporation [3], and exonuclease proofreading reactions [6] These papers provided valuable insights into the kinetic basis for the high DNA replication fidelity of T7 DNA polymerase but were flawed in the analysis of data regarding a prechemistry nucleotide-induced conformational change, based on the interpretation of an observed small thio-elemental effect. The first direct measurement of a prechemistry conformational change came from studies on the low-fidelity repair enzyme, polymerase b, using a 2-aminopurine fluorescent reporter in the DNA [9,10,11] These studies showed that the conformational change was faster than chemistry, challenging previous notions that the conformational change had to be rate-limiting to contribute to fidelity. Our optimized methods can be extended to other enzyme systems
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