THE MAJOR, N-2-GUA ADDUCT OF THE (+)-ANTI-BENZO[A]PYRENE DIOL EPOXIDE CAN BE UNSTABLE IN DOUBLE-STRANDED DNA

The mechanisms of mutagenesis by the (+)-anti diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] was investigated in supF of the Escherichia coli plasmid pUB3 [Rodriguez and Loechler (1993) Biochemistry 32, 1759]. pUB3 was reacted with (+)-anti-B[a]PDE, then either (1) transformed immediately into E. coli or (2) heated at 80 degrees C for 10 min prior to transformation-the latter to probe mechanism. Qualitatively, heating did not have a statistically significant effect on the mutagenic pattern, except at the major base substitution hot spot, G(115), in supF; principally, G(115) --> T mutations were obtained prior to heating, while after heating, G(115) --> A and G(115) --> C mutations became more prevalent. Quantitatively, heating caused an similar to 2-fold decrease in mutation frequency. Heating released a small fraction of adducts (similar to 5%), and the chemistry and implications of this reaction are investigated herein. Although the major adduct of (+)-anti-B[a]PDE (formed at N-2-Gua) is generally regarded to be heat stable, it can be quite unstable in double-stranded (but not single-stranded) DNA at low [Mg2+]. Heating releases the corresponding tetraols from (+)-anti-B[a]P-N-2-Gua in approximately the same ratio as for simple hydrolysis of (+)-anti-B[a]PDE itself. This and other results suggest that guanine remains in DNA when (+)-anti-B-[a]P-N-2-Gua adducts are hydrolyzed. [No evidence for any other chemical change in (+)-anti-B[a]PDE adducts was found.] While no general acid/base or nucleophilic catalysis was observed, adduct hydrolysis was specific acid catalyzed down to pH similar to 5.6, where the pH-rate profile showed a break to a slope of similar to 0.0. This break probably indicates the pK(a) of (+)-anti-B[a]P-N-2-Gua protonated at the N-2-position, which is higher than expected. If true, it suggests that this adduct can become conformationally strained in double-stranded DNA, thereby disrupting resonance between the N-2-position and the rest of the guanine moiety of the adduct and facilitating hydrolysis by raising the pK(a) at N-2. Although heating causes adduct hydrolysis, various arguments suggest that hydrolysis is probably not the cause of either the quantitative or qualitative changes in mutagenesis. It is more Likely that these mutagenic changes are the consequence of the fact that a single adduct (i.e., (+)-anti-B[a]P-N-2-Gua) can adopt multiple conformations in DNA with different mutagenic consequences.