Key Enzyme TDG Repairs Mutagenic DNA Oxidation Damage Linked to Aging and Cancer

Oxidative stress damages DNA continuously throughout life, producing lesions that cause mutations, cancer, and cellular aging. While much is known about repair of the guanine oxidation product oxoG, the major adenine oxidation lesion—7,8-dihydro-8-oxoadenine (oxoA)—has received far less attention. oxoA is mutagenic in mammalian cells because DNA polymerases insert dGTP opposite it, causing A→C transversions. Understanding how cells repair oxoA is therefore important for understanding cancer and aging biology.

This study by Servius and Drohat systematically characterized the excision of oxoA from DNA by thymine DNA glycosylase (TDG), an enzyme best known for removing thymine from G·T mispairs and for initiating active DNA demethylation. Using single-turnover kinetics across a range of enzyme concentrations, the authors determined three key parameters: maximal activity (k_max), substrate affinity (K_0.5), and catalytic efficiency (k_max/K_0.5) for four oxoA base-pair contexts.

The results reveal enormous variation in TDG's catalytic efficiency depending on the opposing base. G·oxoA pairs are processed with k_max/K_0.5 of ~3,919 μM⁻¹ min⁻¹—extraordinarily high for a DNA glycosylase—while T·oxoA pairs yield only ~0.54 μM⁻¹ min⁻¹, a 7,276-fold difference. This disparity reflects both weaker binding (higher K_0.5) and reduced chemistry (lower k_max) for less favored substrates. The 3′-neighboring base also strongly modulates activity, especially for T·oxoA pairs where a 3′-G is preferred by up to 68-fold over 3′-T, consistent with known TDG sequence preferences for pyrimidine substrates.

A key mechanistic finding concerns how TDG activates departure of the oxoA leaving group. The adenine lesion has a low pKa at N1 (~3), raising the possibility of acid catalysis. However, pH-rate profiles show TDG excision of oxoA is pH-independent between pH 5.5 and 8.5, ruling out acid catalysis. This contrasts with MutY (which uses acid catalysis to remove normal adenine) and with TDG's own acid-catalyzed excision of 5-carboxylcytosine. Instead, TDG appears to stabilize an anionic oxoA leaving group, an unusual catalytic strategy.

The study also dissects the roles of two conserved active-site residues: H151 and Y152. H151 strongly promotes oxoA excision (up to 73-fold effect) but paradoxically antagonizes excision of thymine and uracil, suggesting it plays substrate-specific roles. The hydroxyl group of Y152 is required for efficient excision of oxoA and thymine but is dispensable for uracil, 5-formylcytosine, and 5-carboxylcytosine excision, while the aromatic ring of Y152 is essential for all substrates. These findings reveal unexpected mechanistic diversity within TDG's active site and provide a framework for understanding how a single enzyme can repair a chemically diverse range of DNA lesions.