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Impact of guanine damage on DNA methylation by Dnmt3aтезисы доклада
- Авторы: Gromova E.S., Kirsanova O.V., Sergeev A.V., Minero A.S., Lukashevich O.V., Cherepanova N.A., Kolbanovskiy A., Geacintov N.E.
- Сборник: Materials of The Seventh NEB Meeting on DNA Restriction and Modification, August 24-29, 2015, Gdańsk, Poland
- Тезисы
- Год издания: 2015
- Место издания: Gdańsk, Poland
- Первая страница: 40
- Последняя страница: 41
- Аннотация: Methylation of the CpG sites in DNA is an epigenetic modification of the genomes that plays an important role in regulation of many biologic processes. DNA methyltransferase Dnmt3a is involved in the formation of de novo methylation pattern. DNA is constantly attacked by reactive species generated during cellular metabolism, exposure to environment, and action of therapeutics. Benzo[a]pyrene (B[a]P) is one of the most common polycyclic aromatic hydrocarbons which are carcinogens and environmental pollutants. It is metabolized to highly genotoxic diol epoxides (B[a]PDE) which react chemically with guanine in DNA. 6-thioguanine (SG) is widely used antileukemic agent. The incorporation of B[a]PDE-dG or SG into CpG sites may perturb the cytosine methylation by Dnmt3a. To test this hypothesis we examined site-specifically modified 30-mer DNA containing either stereoisomeric B[a]PDE-dG adducts or SG residues as substrates of catalytic domain of murine Dnmt3a (Dnmt3a-CD). The methylation rates were significantly reduced by trans-B[a]PDE-dG (minor groove adducts) regardless of their position in the substrate and by cis-B[a]PDE-dG (intercalated adducts) within the CpG site. In Dnmt3a-CD/B[a]PDE-DNA complexes the stereochemistry-dependent fluorescence enhancement of the covalently bound B[a]PDE residues was observed. We suggested that the movement of the Dnmt3a-CD catalytic loop and flipping of the target cytosine are disturbed by the bulky B[a]PDE residues when they are in the minor groove. The impact of intercalated adducts arises from the distortion of the CpG site. When SG replaced guanine within the CpG site 3’-adjacent to the target cytosine decrease in methylation was observed. This effect may be due to the violation of the Dnmt3a-CD contact to the 6-oxo group of the guanine which was predicted to be important for the formation of a catalytically competent Dnmt3a-CD/DNA complex (1). SG absorption band is above 300 nm where DNA and the enzyme are transparent. This prompted us to follow the local methylation-associated conformational changes in SG-DNA by circular dichroism above 300 nm using SG as spectroscopic probe. Thus, the disturbance of base stacking in the CpG site within Dnmt3a-CD/SG-DNA complexes was suggested. The other guanine damages such as 8-oxoguanine and O6-methylguanine also have significant effects on the Dnmt3a-catalysed methylation (2,3). Overall, the effect of guanine damages diminishes as follows: 8-oxoG>>(+)-trans-B[a]PDE-dG~(+)-cis-B[a]PDE-dG>O6meG>SG. In summary, our data suggest epigenetic contributions of guanine damages via their impact on the Dnmt3a functioning.
- Добавил в систему: Громова Елизавета Сергеевна