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We have designed an Ha-ras/thymidine kinase (TK) cassette that permits the incorporation of chemically synthesized adducts within specific domains of the rat Ha-ras protooncogene. This cassette has been used to evaluate the mutagenicity of O6-substituted guanine residues, including 06-methylguanine and 06-benzylguanine, incorporated within the 12th codon of this locus. Mutations were monitored by the ability of these modified Ha-ras DNAs to transform Rat4 TKcells. Our results indicate that both types of 06-substituted guanines are substantially mutagenic, although the methyl substituent induced a 2-fold higher percentage of transformed Rat4 TKV colonies than its bulkier benzyl analogue. Interestingly, the mutagenicity of both 0-substituted guanines was found to be independent of their relative position within codon 12, therefore suggesting that the specific activation of Ha-ras oncogenes by GGA -. GAA mutations in tumors induced by methylating carcinogens might be due to differences in the accessibility of these guanine residues to the carcinogen rather than to a differential rate of repair.

Nine 16-base oligodeoxyribonucleotides having the sequence of codons 9 through the first base of codon 14 of the rodent H-ras gene, i.e., 5'-d(GTGGGCGCTG*G*AGGCG)-3', have been synthesized containing either an O6-methyl- (G* = m6G), O6-ethyl- (G* = e6G), or the newly described O6-benzyl-2'-deoxyguanosine residue (G* = b6G) at position 10 and/or 11 from the 5'-end. The conversion of the protected O6-substituted 2'-deoxyguanosine derivatives to the corresponding 3'-[O-(2-cyanoethyl) diisopropylphosphoramidites] and their incorporation into oligodeoxyribonucleotides were conveniently accomplished by using an "in situ" activation approach and automated phosphite triester synthetic methods. These oligomers were characterized by enzymatic digestion to their component nucleosides and were shown to be free of detectable contamination by known nucleoside impurities that can be produced during these syntheses. The melting behavior and circular dichroism spectra are described for duplexes of the nine O6-substituted 2'-deoxyguanosine containing oligomers paired with the complementary strand 5'-d(CGCCTCCAGCGCCCAC)-3', and these data have been compared with those for the "wild-type" unsubstituted duplex.

Product distributions were determined for reactions between 2'-deoxyguanosine, or its anion, and 7-(bromomethyl)benz[a]anthracene in either acetone/H2O (1:1) or 2,2,2-trifluoroethanol at 50 or 70 degrees C. The exocyclic amino-substituted product, N2-(benz[a]anthracen-7-ylmethyl)-2'-deoxyguanosine, was always the major nucleoside product formed in these reactions, although its yield was higher in reactions involving 2'-deoxyguanosine anion than the neutral nucleoside. Reaction with the anion also led to formation of the 1-substituted 2'-deoxyguanosine and a guanidinoimidazole nucleoside resulting from reaction of 2'-deoxyguanosine at carbon 5. Reactions of 2'-deoxyguanosine anion in 2,2,2-trifluoroethanol are shown to produce significant amounts of the N2-substituted product, which is difficult to prepare by other routes.

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