Deamination of 5-methylcytosine to thymine creates mutagenic G·T mispairs, contributing to cancer and genetic disease. Thymine DNA glycosylase (TDG) removes thymine from these G·T lesions, and follow-on base excision repair yields a G·C pair. A previous crystal structure revealed TDG (catalytic domain) bound to abasic DNA product in a 2:1 complex, one subunit at the abasic site and the other bound to undamaged DNA. Biochemical studies showed TDG can bind abasic DNA with 1:1 or 2:1 stoichiometry, but the dissociation constants were unknown, as was the stoichiometry and affinity for binding substrates and undamaged DNA. We showed that 2:1 binding is dispensable for G·U activity, but its role in G·T repair was unknown. Using equilibrium binding anisotropy experiments, we show that a single TDG subunit binds very tightly to G·U mispairs and abasic (G·AP) sites, and somewhat less tightly G·T mispairs. Kinetics experiments show 1:1 binding provides full G·T activity. TDG binds undamaged CpG sites with remarkable affinity, modestly weaker than G·T mispairs, and exhibits substantial affinity for nonspecific DNA. While 2:1 binding is observed for large excess TDG concentrations, our findings indicate that a single TDG subunit is fully capable of locating and processing G·U or G·T lesions.
Bibliographical noteFunding Information:
The National Institutes of Health (R01-GM-072711). Funding for open access charge: National Institutes of Health, USA.