1,N2-Ethenoguanine, a Mutagenic DNA Adduct, Is a Primary Substrate of Escherichia coliMismatch-specific Uracil-DNA Glycosylase and Human Alkylpurine-DNA-N-Glycosylase

The promutagenic and genotoxic exocyclic DNA adduct 1,N2-ethenoguanine (1,N2-εG) is a major product formed in DNA exposed to lipid peroxidation-derived aldehydes in vitro. Here, we report that two structurally unrelated proteins, theEscherichia coli mismatch-specific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N-glycosylase (ANPG), can release 1,N2-εG from defined oligonucleotides containing a single modified base. A comparison of the kinetic constants of the reaction indicates that the MUG protein removes the 1,N2-εG lesion more efficiently (kcat/Km = 0.95 × 10−3 min−1 nm−1) than the ANPG protein (kcat/Km = 0.1 × 10−3 min−1 nm−1). Additionally, while the nonconserved, N-terminal 73 amino acids of the ANPG protein are not required for activity on 1,N6-ethenoadenine, hypoxanthine, orN-methylpurines, we show that they are essential for 1,N2-εG-DNA glycosylase activity. Both the MUG and ANPG proteins preferentially excise 1,N2-εG when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N2-εG when it is opposite dG. Using cell-free extracts from genetically modified E. coli and murine embryonic fibroblasts lacking MUG and mANPG activity, respectively, we show that the incision of the 1,N2-εG-containing duplex oligonucleotide has an absolute requirement for MUG or ANPG. Taken together these observations suggest a possible role for these proteins in counteracting the genotoxic effects of 1,N2-εG residues in vivo.