Rapid excision of oxidized adenine by human thymine DNA glycosylase

Oxidation of DNA bases generates mutagenic and cytotoxic lesions that are implicated in cancer and other diseases. Oxidative base lesions, including 7,8-dihydro-8-oxoguanine, are typically removed through base excision repair. In addition, oxidized deoxynucleotides such as 8-oxo-dGTP are depleted by sanitizing enzymes to preclude DNA incorporation. While pathways that counter threats posed by 7,8-dihydro-8-oxoguanine are well characterized, mechanisms protecting against the major adenine oxidation product, 7,8-dihydro-8-oxoadenine (oxoA), are poorly understood. Human DNA polymerases incorporate dGTP or dCTP opposite oxoA, producing mispairs that can cause A→C or A→G mutations. oxoA also perturbs the activity of enzymes acting on DNA and causes interstrand crosslinks. To inform mechanisms for oxoA repair, we characterized oxoA excision by human thymine DNA glycosylase (TDG), an enzyme known to remove modified pyrimidines, including deaminated and oxidized forms of cytosine and 5-methylcystosine. Strikingly, TDG excises oxoA from G⋅oxoA, A⋅oxoA, or C⋅oxoA pairs much more rapidly than it acts on the established pyrimidine substrates, whereas it exhibits comparable activity for T⋅oxoA and pyrimidine substrates. The oxoA activity depends strongly on base pairing and is 370-fold higher for G⋅oxoA versus T⋅oxoA pairs. The intrinsically disordered regions of TDG contribute minimally to oxoA excision, whereas two conserved residues (N140 and N191) are catalytically essential. Escherichia coli mismatch-specific uracil DNA-glycosylase lacks significant oxoA activity, exhibiting excision rates 4 to 5 orders of magnitude below that of its ortholog, TDG. Our results reveal oxoA as an unexpectedly efficient purine substrate for TDG and underscore the large evolutionary divergence of TDG and mismatch-specific uracil DNA-glycosylase.