REACTION OF DNA WITH ALKYLATING-AGENTS - DIFFERENTIAL ALKYLATION OF POLY[DA-DT] BY METHYLNITROSOUREA AND ETHYLNITROSOUREA

The poly[dA-dT] system developed to study the reaction of the alkylating species generated by ethylnitrosourea with nucleophilic sites on A-T base pairs in the helical β conformation of DNA (Jensen, D. E. & Reed, D. J. (1978) Biochemistry 17 (preceding paper in this issue)) is extended to consider the methylation of this polymer resulting from incubation with methylnitrosourea. The assay for the kinds and degree of modification involves enzymic hydrolysis of the polymer and resolution of the digestion products utilizing anion-exchange high pressure liquid chromatographic methods. Modification of the phosphate group oxygen atoms using methylnitrosourea is observed to be 65-75% of the total assayed alkylation under a variety of reaction conditions. Some reaction preference for one of the available nucleotide phosphate oxygen sites relative to the other is indicated as determined by the differential yield of methyl dideoxynucleoside phosphotriester diastereomers. A high level of methylation at the 3-position nitrogen atom on the adenine moiety is observed, amounting to approximately 25% of the total alkylation. A low degree of methylation occurs at the 2-position and 4-position oxygen sites on thymine, the O2-thymine modification accounting for only 3-5% of the total methylation products assayed. These results are in distinct contrast to our findings using ethylnitrosourea as the alkylating agent. A reaction mechanism to account for these differences is discussed. © 1978, American Chemical Society. All rights reserved.