The Major, N2-Gua Adduct of the (+)-anti-Benzo[a]pyrenediol Epoxide Can Be Unstable in Double-Stranded DNA

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Abstract
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 & 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, G115, in supF; principally, G115-->T mutations were obtained prior to heating, while after heating, G115-->A and G115-->C mutations became more prevalent. Quantitatively, heating caused an approximately 2-fold decrease in mutation frequency. Heating released a small fraction of adducts (approximately 5%), and the chemistry and implications of this reaction are investigated herein. Although the major adduct of (+)-anti-B[a]PDE (formed at N2-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-N2-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-N2-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 approximately 5.6, where the pH-rate profile showed a break to a slope of approximately 0.0. This break probably indicates the pKa of (+)-anti-B[a]P-N2-Gua protonated at the N2-position, which is higher than expected.(ABSTRACT TRUNCATED AT 250 WORDS)

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