Substrate specificity of human thymine-DNA glycosylase on exocyclic cytosine adducts

The environmental carcinogen glycidaldehyde (GDA) and therapeutic chloroethylnitrosoureas (CNUs) can form hydroxymethyl etheno and ring-saturated ethano bases, respectively. The mutagenic potential of these adducts relies on their miscoding properties and repair efficiency. In this work, the ability of human thymine-DNA glycosylase (TDG) to excise 8-(hydroxymethyl)-3,N4-ethenocytosine (8-hm-ɛC) and 3,N4-ethanocytosine (EC) was investigated and compared with ɛC, a known substrate for TDG. When tested using defined oligonucleotides containing a single adduct, TDG is able to excise 8-hm-ɛC but not EC. The 8-hm-ɛC activity mainly depends on guanine pairing with the adduct. TDG removes 8-hm-ɛC less efficiently than ɛC but its activity can be significantly enhanced by human AP endonuclease 1 (APE1), a downstream enzyme in the base excision repair. TDG did not show any detectable activity toward EC when placed in various neighboring sequences, including the 5′-CpG site. Molecular modeling revealed a possible steric clash between the non-planar EC exocyclic ring and residue Asn 191 within the TDG active site, which could account for the lack of TDG activity toward EC. TDG was not active against the bulkier exocyclic adduct 3,N4-benzethenocytosine, nor the two adenine derivatives with same modifications as the cytosine derivatives, 7-hm-ɛA and EA. These findings expand the TDG substrate range and aid in understanding the structural requirements for TDG substrate specificity.