We have evaluated conditions in solution which favor the deamination of cytosine and its analogues in order to understand factors which might induce in vivo spontaneous GC to AT base transition mutations in DNA. We hypothesize that the deamination reaction is kinetically enhanced when the cytosine is part of a mismatched base pair or is juxtaposed opposite certain modified bases, including 06-alkyl guanine and 2-aminopurine. Our proposed experiments are directed at showing that certain reagents which are known carcinogens will considerably enhance the probability of deamination of a cytosine or 5-methylcytosine residue, and that """"""""cross-strand protonatin"""""""" may be a likely mechanism for carcinogen-enhanced spontaneous mutagenesis. In our model, carcinogens resulting from the reactions of nitrosoureas and other alkylating agents that form 06-guanine derivatives would greatly facilitate the spontaneous deamination of a cytosine or 5-methylcytosine residue situated nearby in the helix. Cross-strand induced mutagenicity in M13 double stranded DNAs containing alkylated, damaged or mispaired bases will be analyzed with a site-specific mutagenesis assay developed during the current grant period. Structural similarities between the potent mutagen 2-aminopurine and the reaction products derived upon chemical alkylation of guanine make it possible for us to propose a general pathway for spontaneous mutagenesis induced by many alkylating agents and by 2-aminopurine. We will utilize 2-aminopurine and 06-alkyl guanine in DNA polymers as model systems for testing our hypothesis. Nuclear magnetic resonance (NMR) and calorimetry will be used to characterize the hydrogen bond interactions of pairs and mispairs of nucleic acid bases. We predict that 5-methylcytosine would deaminate more quickly than cytosine in vivo, consistent with in vitro results, and that certain carcinogens would drastically accelerate the deamination of cytosine and 5-methylcytosine with conversion to uracil and thymine respectively.
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