Implications of cytosine methylation on (+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide N-2-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 (a nti-BPDE) to the N-2-amino group of deoxyguanine in the oligonucleotides 5' -d(CCTATCGXTATCC) and 5'-d(CCTATm(5)CGXTATCC) (X being T, A, or C) has been studied. The extent of formation of the (+)-trans-anti-BPDE-N-2-dG adduct in single-stranded 13-mer oligonucleotides with 5'-d(m(5)-CGT) and 5'-d(m(5 )CGA) sequence contexts was significantly higher (1.5- and 2.4-fold, respec tively) relative to that of the nonmethylated sequences. With the 5'-d(CGC) sequence context, m(5)dC had no significant effect on adduct formation. Wh en the reaction was allowed to proceed in the presence of oligonucleotide d uplexes (composed of a 13-mer parent strand and a 9-mer complement), a sign ificant increase in the extent of adduct formation was observed with 5'-d(m (5)CGT)/d(CGA) and 5'-d(m(5)CGA)id(CCT), but not with 5'-d(CGC)/d(GCG), rel ative to those of the nonmethylated duplexes. Independent of sequence conte xt, no clear effect of m(5)dC on diol epoxide binding to the opposite dG in the complementary strand was observed. The level of diol epoxide binding t o the dG target-in the 13-mer oligonucleotides is in general higher in sing le-stranded sequences than in the duplexes. With 5'-d(CGA) and 5'-d(m(5)CGA ), for instance, adduct yields were 3- and 4-fold higher, respectively. The thermodynamic stability of the (+)-trans-anti-BPDE-N-2-dG adduct in the 5' -d(m(5)CGT)-containing duplex (composed of a 13-mer parent strand and a ful l complement) was substantially higher than in the 5'-d(CG-T)/d(GCA) sequen ce context. The stimulating effect-of cytosine methylation on the formation of DNA adducts of anti-BPDE has previously been demonstrated in other expe rimental systems. The increase in yield could possibly be rationalized in t erms of prestacking of the pyrenyl ring system with the nucleobases prior t o the nucleophilic addition reaction of the exocyclic amino group. The resu lts from induced circular dichroism studies with the (+)-trans-anti-BPDE-N- 2-dG adduct in the 5'-d(m(5)CGT)-containing duplex are consistent with subs tantial heterogeneity of adduct conformations, including both external mino r groove-localized and intercalated structures.