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 coil 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 pri or to transformation-the latter to probe mechanism. Qualitatively, hea ting did not have a statistically significant effect on the mutagenic pattern, except at the major base substitution hot spot, G(115), in su pF; 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 de crease in mutation frequency. Heating released a small fraction of add ucts (similar to 5%), and the chemistry and implications of this react ion 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 c an be quite unstable in double-stranded (but not single-stranded) DNA at low [Mg2+]. Heating releases the corresponding tetraols from (+)-an ti-B[a]P-N-2-Gua in approximately the same ratio as for simple hydroly sis of (+)-anti-B[a]PDE itself. This and other results suggest that gu anine remains in DNA when (+)-anti-B-[a]P-N-2-Gua adducts are hydrolyz ed. [No evidence for any other chemical change in (+)-anti-B[a]PDE add ucts 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 o f 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 exp ected. If true, it suggests that this adduct can become conformational ly strained in double-stranded DNA, thereby disrupting resonance betwe en the N-2-position and the rest of the guanine moiety of the adduct a nd facilitating hydrolysis by raising the pK(a) at N-2. Although heati ng causes adduct hydrolysis, various arguments suggest that hydrolysis is probably not the cause of either the quantitative or qualitative c hanges 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 m utagenic consequences.