NITROSAMINE-INDUCED CANCER - O4-ALKYLTHYMINE PRODUCES SITES OF DNA HYPERFLEXIBILITY

The carcinogenic properties of N-nitroso compounds are associated with their ability to alkylate DNA, in particular to form O6-alkylguanine and O4-alkylthymine. DNA duplexes containing either O6-alkylguanine or O4-alkylthymine were synthesized, and each duplex was ligated to form a set of DNAs of increasing length with the alkylated base out of phase (16 base-pairs apart) or in phase (21 base-pairs apart) with the helical repeat of the DNA. The DNA contained the sequence 5' CAA 3', which is the 6 1 st codon of the K-ras gene, because this codon is a preferred site of mutation for a number of carcinogens including the methylating carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). O4-Methylthymine or O4-ethylthymine replaced thymine in either of the two A.T base-pairs of this codon (normally CAA), and O6-methylguanine replaced the guanine in the G.C pair. All the sequences containing 04-alkylthymine exhibited anomalous, slow, gel migration and ligated to form circles of unusually small diameter. In general, the effect was seen when the alkylated base-pair was out of phase with the helical repeat as well as when it was in phase, suggesting that the alkylated base-pair confers flexibility which is largely isotropic, i.e., has no preferred direction, rather than anisotropic flexibility or bending. However, at pH 8.3 the 21-base-pair set containing O4-alkylT.A had significantly greater anomalous migration than the 16-base-pair set, suggesting that the flexibility produced by this base-pair has a significant anisotropic component and thus resembles true bending. This anisotropic component was eliminated when the pH of the gel was decreased from 8.3 to 6.5. NMR studies have previously suggested that the adenine in this base-pair is protonated at pH 5.5 and that this proton forms a hydrogen bond between N1 of the purine and 02 of the pyrimidine. Protonation of adenine at pH 6.5 (but not at 8.3) and the consequent formation of a second hydrogen bond in the base-pair may explain the difference in electrophoretic behavior at the different pHs. DNA duplexes containing O4-alkylT.A base-pairs were more retarded and had lower thermal melting points (T(m)) than DNA duplexes containing O4-alkylT.G base-pairs. The effect of O4-methylthymine was much greater than that of O6-methylguanine, and the effect of O4-ethylthymine was slightly greater than that of O4-methylthymine.