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DNA sequence dependence of guanine-O alkylation by the N-nitroso carcinogens N-methyl- and N-ethyl-N-nitrosourea
Authors:Kirsten Sendowski  Manfred F. Rajewsky  
Abstract:
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 gunine-O6 alkylation does nor 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-(O6-2′-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 5t́ and 3′ sides. Thus, a 5′ neighboring guanine, followed by 5t́ 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-methylcytocine in the 5′ position being only slightly less inhibitory). Thymine and cytosine were more ‘permissive’ when placed 3′ in comparison with their affects in the 5′ flanking position.
Keywords:N-Alkyl-N-nitrosourea   Guanine-O6 alkylation   DNA sequence   Oligonucleotides   Monoclonal antibodies
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