In vivo and in Vitro Replication Consequences of Stereoisomeric Benzo[a]pyrene-7,8-dihydrodiol 9,10-Epoxide Adducts on Adenine N6 at the Second Position of N-ras Codon 61

Abstract

Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo[a]pyrene, is a mutagenic in both bacterial and mammalian systems. Toward understanding the mutagenic effects of different stereoisomers of BPDE at specific sites in DNA, six stereochemically defined BPDE adducts were constructed on adenine N6 at position 2 of the human N-ras 61 codon within an 11-base oligonucleotide fragment. Both the nonadducted and BPDE-adducted N-ras 61 11-mers were inserted into a unique EcoRI site in single-stranded M13mp7L2 DNA and utilized for in vivo studies. The ligation efficiencies of BPDE-adducted 11-mers into the single-stranded vector were determined by Southern hybridization and confirmed by electron microscopy. Repair-deficient AB2480 E. coli cells were transformed with adducted and non-adducted DNA samples. The resultant plaque-forming abilities were used to evaluate the replication competence of the various BPDE adducts with respect to the nonadducted 11-mer. Point mutations due to aberrant replication at the adducted site were identified by the technique of differential DNA hybridization. All of the six BPDE adducts examined were mutagenic in vivo, generating exclusively A-->G mutations at frequencies ranging from 0.26 to 1.20%. In vitro replication studies using these BPDE-adducted 11-mers involved primer extension assays with Klenow fragment. All of the BPDE-modified templates demonstrated distinct blockage at the adducted site and/or 1 base 3' to the adducted site, allowing essentially no translesion synthesis to form fully extended polymerization products in vitro.

Position-specific trapping of topoisomerase I–DNA cleavage complexes by intercalated benzo[ a ]- pyrene diol epoxide adducts at the 6-amino group of adenine

“…Cleavage products containing a trans-S adduct migrated slightly more slowly than did the corresponding fragments containing a trans-R adduct. Differences in electrophoretic migration for oligonucleotides containing R-versus S-BaP DE adducts have been noted previously (31,32).…”

Highly Mutagenic Bypass Synthesis by T7 RNA Polymerase of Site-specific Benzo[a]pyrene Diol Epoxide-adducted Template DNA

“…The stereochemistry of BPDE has an influence on its potent mutagenic and carcinogenic properties (11)(12)(13)(14). It has been shown that BPDE-adducted DNA blocks synthesis by a variety of DNA polymerases (15)(16)(17)(18)(19). Several studies have also demonstrated that BPDE adducts in DNA impede transcription (20 -27).…”

The role of polycyclic aromatic hydrocarbon–DNA adducts in inducing mutations in mouse skin

“…To make a similar correlation with stable adducts, the BPDE-N 6 dA stable adducts should be highly mutagenic and account for the codon 61 A to T mutations. However, the BPDE-N 6 dA adduct does not appear to be exceptionally mutagenic [82] and forms A to G mutations in codon 61 [69].…”

“…Knowing the mutational specificity in the preneoplastic tissue is important, because it can reveal the critical lesions for cancer. In vitro, the mutational specificity is obtained from studies that examine polymerase errors during the filling synthesis of nicks or gaps similar to those generated by the excision steps of NER and BER [66,67], or those during bypass over PAH-DNA stable adducts (translesion synthesis), similar to those left unrepaired by NER [68,69]. The knowledge obtained from such studies is limited to the specific DNA polymerases, and sometimes cannot explain mutagenesis in vivo.…”

“…For example, mouse skin papillomas induced by benzo[a]pyrene (BP) show both codon 13 (GGC to GTC) and 61 (CAA to CTA) mutations in the H-ras gene [16]. Translesional synthesis errors over BP diolepoxide (BPDE)-N 2 dG adducts at the central G of GGC form the GTC mutation [49], but the BPDE-N 6 dA adducts do not form the CTA mutation at the central A of CAA [69,70].…”