Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods

The impact of bulky carcinogen-DNA adducts positioned at or near recognition sites (CpG) of eukaryotic DNA methyltransferases on their catalytic activities is poorly understood. In the present study, we employed site-specifically modified 30-mer oligodeoxyribonucleotides containing stereoisomeric benzo[a]pyrene diol epoxide (B[a]PDE)-derived guanine (B[a]PDE-N²-dG) or adenine (B[a]PDE-N⁶-dA) adducts of different conformations as substrates of the catalytic domain of murine Dnmt3a (Dnmt3a-CD). The fluorescence of these lesions was used to examine interactions between Dnmt3a-CD and DNA. In B[a]PDE-DNA¢Dnmt3a-CD complexes, the intensity of fluorescence of the covalently bound B[a]PDE residues is enhanced relative to the protein-free value when the B[a]PDE is positioned in the minor groove [(+)- and (-)-trans-B[a]PDE-N²-dG adducts in the CpG site] and when it is intercalated on the 5'-side of the CpG site [(+)-trans-B[a]PDE- N⁶-dA adduct]. The fluorescence of B[a]PDE-modified DNA¢Dnmt3a-CD complexes exhibits only small changes when the B[a]PDE is intercalated with base displacement in (+)- and (-)-cis-B[a]PDE-N²-dG adducts and without base displacement in the (-)-trans-B[a]PDE-N⁶-dA adduct. The initial rates of methylation were significantly reduced by the minor groove trans-B[a]PDE-N²-dG adducts, regardless of their position in the substrate and by the intercalated cis-B[a]PDE-N²-dG adducts within the CpG site. The observed changes in fluorescence and methylation rates are consistent with the flipping of the target cytosine and a catalytic loop motion within the DNA¢Dnmt3a-CD complexes. In the presence of the regulatory factor Dnmt3L, an enhancement of both methylation rates and fluorescence was observed, which is consistent with a Dnmt3L-mediated displacement of the catalytic loop towards the CpG site.