Steady-state kinetic analysis of DNA polymerase single-nucleotide incorporation products

Abstract

This unit describes the experimental procedures for the steady-state kinetic analysis of DNA synthesis across DNA nucleotides (native or modified) by DNA polymerases. In vitro primer extension experiments with a single nucleoside triphosphate species followed by denaturing polyacrylamide gel electrophoresis of the extended products is described. Data analysis procedures and fitting to steady-state kinetic models is presented to highlight the kinetic differences involved in the bypass of damaged versus undamaged DNA. Moreover, explanations concerning problems encountered in these experiments are addressed. This approach provides useful quantitative parameters for the processing of damaged DNA by DNA polymerases.

Keywords: DNA polymerase; steady-state kinetics; translesion synthesis.

Figure 1

Chemical structures of (A) thymidine (T), (B) O⁴-methylthymidine (O⁴MeT) inserts, (C) 5’-(6-carboxyfluorescein)-thymidine ((FAM)T) and (D) sequences of DNA template and primer indicating positions of the modification (X) and (FAM)T.

Figure 2

18% (19:1, w/w) Denaturing PAGE analysis of the extension reaction screen by hpol η inserting dATP across from O⁴MeT. hpol η concentrations are reported on the left, reaction time at the top, dNTP concentration at the bottom, and band identity at the right.

Figure 3

18% (19:1, w/w) Denaturing PAGE analysis of the extension reaction (5 min) by hpol η (2.5 nM) across O⁴MeT with varying dATP. dNTP concentrations are reported at the bottom and band identity at the right.

Figure 4

Steady-state kinetic analysis of dATP incorporation by hpol η (v (min⁻¹) versus [dATP] (plotted using Graphpad® Prism software).