Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid.

ABSTRACT: High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of differen

SUBMITTER: Dangerfield TL 

PROVIDER: S-EPMC8715121 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature