Abstract
The promutagenic and genotoxic exocyclic DNA adduct 1,N(2)-ethenoguanine (1,N(2)-epsilonG) is a major product formed in DNA exposed to lipid peroxidation-derived aldehydes in vitro. Here, we report that two structurally unrelated proteins, the Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N-glycosylase (ANPG), can release 1,N(2)-epsilonG 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,N(2)-epsilonG lesion more efficiently (k(cat)/K(m) = 0.95 x 10(-3) min(-1) nm(-1)) than the ANPG protein (k(cat)/K(m) = 0.1 x 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,N(6)-ethenoadenine, hypoxanthine, or N-methylpurines, we show that they are essential for 1,N(2)-epsilonG-DNA glycosylase activity. Both the MUG and ANPG proteins preferentially excise 1,N(2)-epsilonG when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N(2)-epsilonG 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,N(2)-epsilonG-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,N(2)-epsilonG residues in vivo.
Highlights
Ʈ Supported by a postdoctoral fellowship from the Fondation pour la Recherche Medicale
When the various E. coli and human DNA glycosylases were tested on the ⑀G311⁄7C (Fig. 2), only incubation with mismatchspecific uracil-DNA glycosylase (MUG), ANPG70, or APDG led to the cleavage of the labeled oligonucleotide at the position of the modified base
The results presented show that the E. coli MUG and ANPG proteins excise 1,N2-⑀G when present in duplex oligonucleotides
Summary
Ʈ Supported by a postdoctoral fellowship from the Fondation pour la Recherche Medicale. We report that two structurally unrelated proteins, the Escherichia coli mismatchspecific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N-glycosylase (ANPG), can release 1,N2-⑀G from defined oligonucleotides containing a single modified base.
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