Structural basis of damage recognition by thymine DNA glycosylase: Key roles for N-terminal residues

Thymine DNA Glycosylase (TDG) is a base excision repair enzyme functioning in DNA repair and epigenetic regulation. TDG removes thymine from mutagenic G·T mispairs arising from deamination of 5-methylcytosine (mC), and it processes other deamination-derived lesions including uracil (U). Essential for DNA demethylation, TDG excises 5-formylcytosine and 5-carboxylcytosine, derivatives of mC generated by Tet (ten-eleven translocation) enzymes. Here, we report structural and functional studies of TDG(82-308), a new construct containing 29 more N-terminal residues than TDG(111-308), the construct used for previous structures of DNA-bound TDG. Crystal structures and NMR experiments demonstrate that most of these N-terminal residues are disordered, for substrate- or product-bound TDG(82-308). Nevertheless, G·T substrate affinity and glycosylase activity of TDG(82-308) greatly exceeds that of TDG(111-308) and is equivalent to full-length TDG. We report the first high-resolution structures of TDG in an enzyme-substrate complex, for G·U bound to TDG(82-308) (1.54 Å) and TDG(111-308) (1.71 Å), revealing new enzyme-substrate contacts, direct and water-mediated. We also report a structure of the TDG(82-308) product complex (1.70 Å). TDG(82-308) forms unique enzyme–DNA interactions, supporting its value for structure-function studies. The results advance understanding of how TDG recognizes and removes modified bases from DNA, particularly those resulting from deamination.