Uracil-DNA glycosylase efficiency is modulated by substrate rigidity.

Abstract

DNA sequence effects have been identified for many glycosylases and alkyl-transferases that repair base mismatches, uracil bases, alkylated bases, etc. Here, we focused on understanding how DNA sequence impacts the repair of uracil, a highly mutagenic and common lesion in DNA. Uracil DNA-glycosylase (UNG) is a base excision repair enzyme that removes uracil from DNA by a base flipping mechanism. UNG excision efficiency depends on DNA sequence. 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. Time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations of the bare DNA indicated significant differences in substrate flexibilities. We observed a strong correlation between UNG efficiency and substrate flexibility, 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. A better understanding of the fundamental principles that dictate UNG activity has broad implications in diverse fields, from cancer to evolution.