Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods
| Autor: | Antonio S. Minero, Nicholas E. Geacintov, Natalia A. Cherepanova, Olga V. Lukashevich, Elizaveta S. Gromova, Alexander Kolbanovskiy |
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| Rok vydání: | 2012 |
| Předmět: |
Guanine
Stereochemistry Biochemistry Article DNA Methyltransferase 3A Substrate Specificity Adduct Cytosine DNA Adducts Mice chemistry.chemical_compound Catalytic Domain Benzo(a)pyrene Animals heterocyclic compounds DNA (Cytosine-5-)-Methyltransferases Binding site Molecular Biology Binding Sites Base Sequence Adenine Stereoisomerism DNA Cell Biology Methylation DNA Methylation musculoskeletal system Molecular biology Kinetics enzymes and coenzymes (carbohydrates) Spectrometry Fluorescence Oligodeoxyribonucleotides chemistry CpG site Biocatalysis Nucleic Acid Conformation sense organs Protein Binding circulatory and respiratory physiology |
| Zdroj: | FEBS Journal. 279:3965-3980 |
| ISSN: | 1742-464X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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