Pre-Steady-State Kinetic Studies of Nucleotide Incorporation into a Single-Nucleotide Gapped DNA Substrate Catalyzed by Human DNA Polymerase β

Abstract

DNA polymerase β (Polβ) is a key enzyme in DNA base excision repair (BER). Despite extensive research, several microscopic rate constants within the kinetic mechanism of nucleotide incorporation into single-nucleotide gapped DNA by Polβ have not been determined and the identity of the rate-limiting step remains controversial. Here, we employed pre-steady-state kinetic methods and determined the rate constants for correct dNTP association (k₂ = 4.5 × 10⁶ M^–1 s^–1) and dissociation (k_–2 = 118 s^–1) as well as DNA product release (k₇=0.93 s^–1). Previously, uncertainty regarding the transition state of phosphodiester bond formation has led to confusion regarding the interpretation of the sulfur elemental effect between the incorporations of dNTP and its thio analog S_p-dNTPαS. However, recent results from time-resolved X-ray crystallographic studies of three DNA polymerases have allowed us to revise the benchmark of sulfur elemental effect for a rate-limiting chemistry step from 4–11 to 10–160. By using the revised benchmark, we determined the sulfur elemental effects for correct and incorrect nucleotide incorporation to be 3.94 and 64.6, respectively. These suggest the chemistry step limits mismatched, but not matched, nucleotide incorporation. Furthermore, the 2.1-fold difference in the reaction amplitudes of the pulse-quench and pulse-chase assays provides definitive evidence that a protein conformational change step prior to the chemistry step is rate-limiting for matched nucleotide incorporation. These findings unify the kinetic mechanism of correct nucleotide incorporation for Polβ and all other kinetically characterized DNA polymerases and reverse transcriptases, in which the protein conformational change prior to the chemistry step is rate-limiting.