| First Author | Minero AS | Year | 2012 |
| Journal | FEBS J | Volume | 279 |
| Issue | 20 | Pages | 3965-80 |
| PubMed ID | 22913541 | Mgi Jnum | J:202731 |
| Mgi Id | MGI:5521277 | Doi | 10.1111/j.1742-4658.2012.08756.x |
| Citation | Minero AS, et al. (2012) Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods. FEBS J 279(20):3965-80 |
| abstractText | 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-N(2)-dG) or adenine (B[a]PDE-N(6)-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-N(2)-dG adducts in the CpG site] and when it is intercalated on the 5'-side of the CpG site [(+)-trans-B[a]PDE-N(6)-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-N(2)-dG adducts and without base displacement in the (-)-trans-B[a]PDE-N(6)-dA adduct. The initial rates of methylation were significantly reduced by the minor groove trans-B[a]PDE-N(2)-dG adducts, regardless of their position in the substrate and by the intercalated cis-B[a]PDE-N(2)-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. |
