Uracil-DNA glycosylase efficiency is modulated by substrate rigidity

Abstract

Uracil DNA-glycosylase (UNG) is a base excision repair enzyme that removes the highly mutagenic uracil lesion from DNA by a base flipping mechanism. UNG excision efficiency depends on DNA sequence, yet the underlying principles that dictate UNG substrate specificity have remained elusive. Here, we show that UNG efficiency is dictated by the intrinsic local deformability of the substrate sequence around the uracil. UNG specificity constants (kcat/KM) and DNA flexibilities were measured for an engineered set of DNA substrates containing AUT, TUA, AUA, and TUA motifs. Time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations of the bare DNA indicated significant differences in substrate flexibilities. A strong correlation between UNG efficiency and substrate flexibility was observed, with higher kcat/KM values measured for more flexible strands. DNA bending and base flipping were observed in simulations, with more frequent uracil flipping observed for the more bendable sequences. Experiments show that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and resultant UNG activity. The finding that substrate flexibility controls UNG efficiency has implications in diverse fields, including the genesis of mutation hotspots, molecular evolution, and understanding sequence preferences of emerging base editors.