Implications of Cytosine Methylation on (+)-anti-Benzo[a]pyrene 7,8-Dihydrodiol 9,10-Epoxide N2-dG Adduct Formation in 5-d(CGT), 5-d(CGA), and 5-d(CGC) Sequence Contexts of Single- and Double-Stranded Oligonucleotides

Covalent binding of(+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide (anti-BPDE) to the N2-amino group of deoxyguanine in the oligonucleotides 5ʹ-d(CCTATCGXTATCC) and 5ʹd(CCTATm5CGXTATCC) (X being T, A, or C) has been studied. The extent of formation of the (+)-trans-anti-BPʹDE-N^-dG adduct in single-stranded 13-mer oligonucleotides with 5ʹ-d(m5CGT) and 5ʹ-d(m5CGA) sequence contexts was significantly higher (1.5- and 2.4-fold, respectively) relative to that of the nonmethylated sequences. With the 5ʹ-d(CGC) sequence context, m5dC had no significant effect on adduct formation. When the reaction was allowed to proceed in the presence of oligonucleotide duplexes (composed of a 13-mer parent strand and a 9-mer complement), a significant increase in the extent of adduct formation was obseved with 5ʹd(m5CGT)/d(CGA) and 5ʹ-d(m5CGA)/d(CGT), but not with 5ʹ-d(CGC)/d(GCG), relative to those of the nonmethylated duplexes. Independent of sequence context, no clear effect of m5dC on diol epoxide binding to the opposite dG in the complementary strand was observed. The level ofdiol epoxide binding to the dG target in the 13-mer oligonucleotides is in general higher in single-stranded sequences than in the duplexes. With 5ʹ-d(CGA) and 5ʹ-d(m^CGA), for instance, adduct yields were 3- and 4-fold higher, respectively. The thermodynamic stability of the (+)trans-anti-BPDE-N2-dG adduct in the 5ʹ-d(m5CGT)-contaming duplex (composed of a 13-mer parent strand and a full complement) was substantially higher than in the 5ʹ-d(CGT)/d(GCA) sequence context. The stimulating effect of cytosine methylation on the formation of DNA adducts of anti-BPDE has previously been demonstrated in other experimental systems. The increase in yield could possibly be rationalized in terms of prestacking of the pyrenyl ring system with the nucleobases prior to the nucleophilic addition reaction of the exocyclic amino group. The results from induced circular dichroism studies with the (+)-trans-anti-BPDEN2-dG adduct in the 5ʹ-d(m5CGT)-containing duplex are consistent with substantial heterogeneity of adduct conformations, including both external minor groove-localized and intercalated structures.