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Over 80% of ethylnitrosourea and ethylnitrosoguanidine modification of nucleic acids is on oxygens. The reactivity of oxygens (other than ribose and phosphate) in single-stranded RNA is: O2 of C greater than O2 of U greater than O6 of G greater than O4 of U. In double-stranded DNA the order is: O2 of T equals O6 of G greater than O4 of T greater than O2 of C. Oxygen reactivity of single-stranded DNA resembles RNA. The glycosidic bond of O2-alkylpyrimidines is labilised.

0(-Methylguanine (m6G) was incorporated site-specifically into two 25-base oligonucleotides differing only in the nucleotide on the 3' side of the modified base. Templates were primed with oligonucleotides terminating one or two bases prior to the site at which incorporation kinetics were to be investigated. Escherichia coli DNA polymerase I (Klenow fragment) was used to determine the apparent Km and relative V,,x of incorporation of either dCTP or dTTP opposite m6G or G.These data were used to calculate the relative frequency of incorporation opposite the m6G or the unmodified G. When the sequence was 3'-Cm6G-5', there was a 6-to 7-fold preference for formation of a m6G-T pair compared with m6G-C. The m6G-T frequency, based on Vmix/Km, was at least 50-fold greater than that of a G-T pair at the same site. Changing the sequence to 3'-Tm6G-5' had a marked effect on both Km and V.,, of pairs containing m6G and on the incorporation frequency of T opposite m6G, which was then only slightly favored over m6G-C. When replication was started directly opposite m6G, the kinetics appeared unaffected. These data indicate that the frequency of incorporation of C or T opposite m6G in a DNA template is dependent on the flanking neighbors and that a change of even a single base at the 3' position can have a major effect on mutagenic efficiency. Replication using Drosophila Pol a gave the same values for relative frequencies. Pairing of either C or T with m'G on the primer terminus did not significantly inhibit extension of the next normal base pair, in contrast to terminal mismatches of unmodified bases. It is concluded that, in the absence of repair, m6G can exhibit widely differing mutation frequencies which, in these experiments, can be as high as 85% of the replicated base. This variation in frequency of changed pairing could contribute to the occurrence of mutational "hot spots" after replication of damaged DNA.The likely role of 06-alkylguanines as a major factor in mutagenesis by certain alkylating agents was recognized by Loveless about 20 years ago (1). In an attempt to resolve contradictions in the literature, he suggested that O6-methylguanine (m6G) might pair with thymine (T) during replication and thus cause the G-C -* A-T transitions found as the major genetic change after reaction with certain carcinogenic alkylating agents. Further studies in a number of laboratories established that the occurrence, and in particular the persistence of this alkylated base, often correlated with the biological endpoints of mutagenesis and carcinogenesis (2). In accord with the proposed mechanism of mutagenesis, m6G can pair with T both in vitro (3) and in vivo (4), when presented to a polymerase either in the template or as a precursor to DNA synthesis. This model of mutagenesis occurring by base pairing between m6G and T during replication has long been assumed, but only recently has it begun to be tested rigorously [reviewed by Basu and Essigmann (5)]. When an m6G-containing dodecamer was annealed with a series of anal...

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