Sumoylation of thymine DNA glycosylase impairs productive binding to substrate sites in DNA.

The base excision repair enzyme thymine DNA glycosylase (TDG) protects against mutations by removing thymine or uracil from guanine mispairs and functions in active DNA demethylation by excising 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Post-translational modification of TDG by SUMO (small ubiquitin-like modifier) reduces its glycosylase activity but the mechanism remains unclear. We investigated this problem using biochemical and biophysical approaches and a TDG construct comprising residues 82 to 340 (of 410) that includes the SUMOylation site and the motif for non-covalent SUMO binding. Single turnover kinetics experiments were collected at multiple enzyme concentrations ([E]) and the hyperbolic dependence of activity (k _obs ) on [E] yielded the maximal glycosylase activity (k _max ), the enzyme concentration giving half-maximal activity (K _0.5 ), and the catalytic efficiency (k _max /K _0.5 ). Sumoylation of TDG (or TDG ^82-340 ) causes large reductions in catalytic efficiency for G·T, G·U, G·fC, and G·caC DNA substrates, due largely to weakened substrate affinity (increased K _0.5 ). ¹⁹ F NMR experiments show that sumoylation of TDG ^82-340 reduces productive binding to G·U mispairs and dramatically impairs binding to G·T mispairs. A mutation in the TDG SUMO-interacting motif (SIM), E310Q, shown previously to perturb the noncovalent binding of SUMO to unmodified TDG, rescues the glycosylase activity of sumoylated TDG ^82-340 . Similarly, NMR studies show the mutation restores the productive binding of sumoylated TDG ^82-340 to G·U and G·T pairs. Together, the results indicate that intramolecular SUMO-SIM interactions mediate the adverse effect of sumoylation on TDG activity and suggest a model whereby the disruption of SUMO-SIM interactions enables productive binding of sumoylated TDG to substrate sites in DNA.
Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
(Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)