Molecular basis for methoxyamine-initiated mutagenesis: ¹H nuclear magnetic resonance studies of oligonucleotide duplexes containing base-Modified cytosine residues

Methoxyamine, N⁴-methoxycytidine and its 2′-deoxyribo analogue are transition mutagens. The mechanism by which the latter acts after incorporation into or generation within DNA has been ascribed to the ability of the base analogue to pair effectively with both adenine and guanine. To obtain a detailed understanding of these interactions, the solution structures of the self-complementary octanucleotide d(CGGATCCG) and its analogues d(CGGATTCG), d(CGGATMCG) and d(CGGATPCG) (designated 8mer-GC, -GT, -GM and -GP, respectively) were investigated by ¹H nuclear magnetic resonance spectroscopy; M is N⁴-methoxycytosine (mo⁴C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2] oxazin-7-one. A variable temperature study showed the order of stability as 8mer GC > GP > GT > GM. Nuclear Overhauser spectroscopy permitted the assignment of the base, anomeric and H2′/H2″ protons in these 8mers. All had spectra consistent with regular B-DNA duplex structures. Imino proton spectra showed that the 8mers GC, GP and GM involved Watson-Crick base-pairing but that the G · P and to a greater extent G · M base-pairs were in slow exchange on the nuclear magnetic resonance time-scale with the wobble configuration. Indeed, the G · M pair showed an additional exchange process interpreted in terms of the presence of syn and anti conformers of the methoxy group in the wobble pair. This accounts for the destabilization of M compared with the P-containing duplex. The observations are compared with those made earlier on the corresponding AT, AP and AM octamers. It is evident that M and P can form stable base-pairs with both A and G with essentially Watson-Crick geometry. This confirms the earlier, although unsubstantiated explanation for the transition mutational propensity of methoxyamine which, in turn, was based on the fact that methoxycytosine bases have tautomeric constants (K_T) much nearer to unity than the normal bases. The same general explanation for hydroxylamine and hydrazine-induced mutations is correspondingly rendered more certain.