Conformational dynamics during high-fidelity DNA replication and translocation defined using a DNA polymerase with a fluorescent artificial amino acid.

ABSTRACT: We address the role of enzyme conformational dynamics in specificity for a high-fidelity DNA polymerase responsible for genome replication. We present the complete characterization of the conformational dynamics during the correct nucleotide incorporation forward and reverse reactions using stopped-flow and rapid-quench methods with a T7 DNA polymerase variant containing a fluorescent unnatural amino acid, (7-hydroxy-4-coumarin-yl) ethylglycine, which provides a signal for enzyme conformational changes. We show that the forward conformational change (>6000 s^-1) is much faster than chemistry (300 s^-1) while the enzyme opening to allow release of bound nucleotide (1.7 s^-1) is much slower than chemistry. These parameters show that the conformational change sel

SUBMITTER: Dangerfield TL 

PROVIDER: S-EPMC7857513 | biostudies-literature | 2021 Jan-Jun

REPOSITORIES: biostudies-literature