Full Pre-Steady-State Kinetic Analysis of Single Nucleotide Incorporation by DNA Polymerases

By measuring a DNA polymerase incorporation reaction on a very short time scale (5 ms to 10 s) and with a high enzyme concentration, the isolated event of a single nucleotide incorporation can be analyzed. Practically, this is done using a quench-flow instrument, which allows the rapid mixing of the enzyme-primer/template with the nucleotide substrate, after which the reaction is quenched and analyzed. We describe how to titrate the enzyme active site, how to determine, via a scouting experiment, the rate-limiting step in the polymerization reaction, and how to measure the apparent k_pol , K_d(DNA) , and K_d(N) using burst or single-turnover experiments. We include equations for the calculation of the processivity of the polymerase, its nucleotide incorporation specificity and preference, and the activation energy difference for the incorporation of an incorrect nucleotide. Data analysis is discussed, as this is an essential part of accurate data generation in kinetic analyses. © 2019 by John Wiley & Sons, Inc.