Conformational gating governs nucleotide incorporation by a DNA-crosslinked polymerase.

Base excision repair is a major pathway that repairs single-base DNA damage. We recently demonstrated that human DNA polymerase β (hPolβ) fills single-nucleotide gaps after Schiff base formation but before β-elimination, implying that its dRP lyase domain remains covalently crosslinked to DNA during gap-filling synthesis. Because uncrosslinked Polβ dissociates rapidly from DNA (∼3 s-1), mechanistic investigation has been challenging. To elucidate the kinetic mechanism of correct incorporation by DNA-crosslinked hPolβ, we generated a catalytically active crosslinked hPolβ‒DNA complex and performed pre-steady-state kinetic, thermodynamic, and structural analyses. Sulfur elemental effects of 3.7 ± 0.4 and 24 ± 4 for correct and incorrect nucleotide incorporation, respectively, suggest the chemical step is rate-limiting for incorrect, but not for correct, nucleotide incorporation. Pulse-chase and pulse-quench assays revealed a 33% difference in reaction amplitude, establishing the existence of a ternary intermediate preceding the chemical step. Eyring analysis identified a high activation free energy barrier, while the lack of viscosity dependence rules out large domain motions, indicating that the rate-limiting pre-chemical step involves local active-site rearrangements. Together with structurally characterized intermediates, these findings establish the first minimal kinetic mechanism for correct nucleotide incorporation by a DNA-crosslinked polymerase and identify local active-site rearrangements as the rate-limiting step.