Influence of the R(61,2)- and S(61,2)-alpha-(N-6-adenyl)styrene oxide adducts on the A center dot C mismatched base pair in an oligodeoxynucleotide containing the human N-ras codon 61

Conformational studies of R- and S-alpha-(N-6-adenyl)styrene oxide adducts mismatched with deoxycytosine at position X-6 in d(CGGA<(CXA)under bar>GAAG ).d(CTTCTCGTCCG), incorporating codons 60, 61 (underlined), and 62 of the h uman N-ras protooncogene, are described. These were the R- and S(61,2)C add ucts. The S(61,2)C adduct afforded a stable solution structure, while the R (61,2)C adduct resulted in a disordered structure. Distance restraints for the S(61,2)C adduct were calculated from NOE data using relaxation matrix a nalysis. These were incorporated as effective potentials into the total ene rgy equation. The structures were refined using restrained molecular dynami cs calculations which incorporated a simulated annealing protocol. The accu racy of the emergent structures was evaluated by complete relaxation matrix methods. The structures refined to an average rms difference of 1.07 Angst rom, determined by pairwise analysis. The experimentally determined structu re was compared to NOE intensity data using complete relaxation matrix back -calculations, yielding an R-1(x) value of 11.2 x 10(-2). The phenyl ring o f the styrene in the S(61,2)C adduct was in the major groove and remained o riented in the 3'-direction as observed for the corresponding S(61,2) adduc t paired with thymine [Feng, B., Zhou, L., Pasarelli, M., Harris, C. M., Ha rris, T. M., and Stone, M. P. (1995) Biochemistry 34, 14021-14036]. A shift of the modified adenine toward the minor groove resulted in the styrenyl r ing stacking with nucleotide C-5 On the 5'-side of the lesion, which shifte d toward the major groove. Unlike the unmodified A.C mismatch, neither the S(61,2)C nor the R(61,2)C adduct formed protonated wobble A.C hydrogen bond s. This suggests that protonated wobble A.C pairing need not be prerequisit e to low levels of alpha-SO-induced A --> G mutations. The shift of the mod ified adenine toward the minor groove in the S(61,2)C structure may play a more important role in the genesis of A --> G mutations. The disordered str ucture of the R(61,2)C adduct provides a potential explanation as to why th at adduct does not induce A --> G mutations.