DNA sequence dependence of guanine-O6 alkylation by the N-nitroso carcinogens N-methyl- and N-ethyl-N-nitrosourea.

After intracellular in vitro exposure to the mutagenic and carcinogenic N-nitroso compounds N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), respectively, the average relative amounts of the premutational lesion O6-alkylguanine represent about 6% and 8% of all alkylation products formed in genomic DNA. At the level of individual DNA molecules guanine-O6 alkylation does not occur at random; rather, the probability of a substitution reaction at the nucleophilic O6 atom is influenced by nucleotide sequence, DNA conformation, and chromatin structure. In the present study, 5 different double-stranded polydeoxynucleotides and 15 double-stranded oligodeoxynucleotides (24-mers) were reacted with MeNU or EtNU in vitro under standardized conditions. Using a competitive radioimmunoassay in conjunction with an anti-(O6-alkyl-2'-deoxyguanosine) monoclonal antibody, the frequency of guanine-O6 alkylation was found to be strongly dependent on the nature of the nucleotides flanking guanine on the 5' and 3' sides. Thus, a 5' neighboring guanine, followed by 5' adenine and 5' cytosine, provided an up to 10-fold more 'permissive' condition for O6-alkylation of the central guanine than a 5' thymine (with a 5-methylcytosine in the 5' position being only slightly less inhibitory). Thymine and cytosine were more 'permissive' when placed 3' in comparison with their effects in the 5' flanking position.