ИСТИНА

Benzo[a]pyrene (B[a]P) is a ubiquitous environmental pollutant. It is metabolized in vivo to highly genotoxic dihydrodiol epoxides (B[a]PDE) which bind to DNA purines thus contributing to the initiation of tumorigenesis. In order to assess the epigenetic contribution of B[a]PDE in tumorigenesis we employed site-specifically modified oligodeoxyribonucleotide duplexes containing stereoisomeric B[a]PDE-derived guanine (B[a]PDE-N2-dG) or adenine (B[a]PDE-N6-dA) adducts of different conformations as substrates of murine DNA methyltransferase Dnmt3a and its catalytic domain (Dnmt3a-CD). The length of the B[a]P-DNA was varied and a nucleosome with a single B[a]P-modified 145-mer DNA duplex was constructed in order to develop an approach that would be closer to in vivo conditions. The initial rates of methylation by Dnmt3a-CD were significantly reduced by the minor groove trans-B[a]PDE-N2-dG adducts, regardless of their position in the substrate, by the intercalated cis-B[a]PDE-N2-dG adducts within the CpG site and by intercalated (+)-trans-B[a]PDE-N6-dA adducts adjacent to the CpG site. The same rules were observed in the case of full-length enzyme and in the presence of the Dnmt3a regulatory factor, Dnmt3L. The B[a]PDE lesions produced only small effect on Dnmt3a-CD binding to DNA. The B[a]PDE-DNA adduct stereochemistry-dependent fluorescence enhancement of the B[a]PDE residues was observed upon interaction with Dnmt3a-CD. These results suggest that the movement of the Dnmt3a-CD catalytic loop and flipping of the target cytosine are disturbed by the B[a]PDE residues when they are in the DNA minor groove. The impact of intercalated adducts arises from the distortion of the CpG site. The formation of non-productive complexes of Dnmt3a-CD with B[a]P-DNA is also possible. Hence, the introduction of B[a]PDE-lesions into DNA, especially the introduction of the most abundant and poorly repairable trans-B[a]PDE-N2-dG adducts, leads to potential alterations of de novo methylation efficiencies thus contributing to the genotoxic effects associated with DNA adduct-induced mutagenic mechanisms.