AP SITES ARE NOT SIGNIFICANTLY INVOLVED IN MUTAGENESIS BY THE (+)-ANTI DIOL EPOXIDE OF BENZO[A]PYRENE - THE COMPLEXITY OF ITS MUTAGENIC SPECIFICITY IS LIKELY TO ARISE FROM ADDUCT CONFORMATIONAL POLYMORPHISM

In previous work, mutations induced by the (+)-anti diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] were scored in the supF gene of the Escherichia coli plasmid pUB3 [Rodriguez & Loechler (1993) Biochemistry 32, 1759]. pUB3 was reacted with (+)-anti-B[a]PDE and then either (1) transformed immediately into E. coli or (2) heated at 80-degrees-C for 10 min prior to transformation. Heating only released a small fraction of adducts (approximately 5%) and did not significantly affect the mutagenic pattern at most sites in supF. However, at the major base substitution hotspot, G115, principally G-->T mutations (87%) were obtained prior to heating, while after heating, G-->T mutations decreased (45%) and G-->A (21%) and G-->C (33%) mutations became more prevalent. One model for this result is that prior to heating a heat-labile adduct at G115 causes one pattern of mutagenesis, but after heating the labile adduct is hydrolyzed to an apurinic site (AP site), which causes a second mutational pattern. To test this, a role for AP sites generated from labile adducts by heating at 80-degrees-C for 10 min is investigated. It is shown that when plasmid pUB3 contains 22 (+)-anti-B[a]PDE adducts, 0.6% (or fewer) are converted to AP sites as determined in an assay based upon the action of an AP-endonuclease. In a separate line of investigation not involving (+)-anti-B[a]PDE adducts, mutation frequency (MF) per AP site is estimated. (In these experiments, AP sites were introduced into pUB3 by the classic procedure of heating at 70-degrees-C/pH 5.0 to hydrolyze purines. In fact, the majority of mutants induced by this procedure probably arose via cytosine deamination to uracil and not via AP sites, based upon three criteria, including that approximately 75% of the mutations were GC-->AT.) Given the number of AP sites formed from (+)-anti-B[a]PDE adducts and the estimate of MF/AP site, we conclude that < approximately 2% of the 115 base substitution mutations induced in supF by (+)-anti-B[a]PDE can be attributed to AP sites when the adducted plasmid was heated at 80-degrees-C for 10 min prior to transformation. (With the unheated adducted plasmid, this limit is < approximately 1%). This makes it unlikely that AP sites are significantly involved in (+)-anti-B[a]PDE mutagenesis, including at G115, where 29% of base substitution mutants were found. The most likely alternative model for the heat-induced changes in mutational pattern at G115, as well as the complexity of the mutagenic spectra of (+)-anti-B[a]PDE in general, is that a single adduct can adopt multiple conformations, each of which can cause different kinds of mutations, and factors such as heating and DNA sequence context can influence adduct conformation.