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Murine DNA methyltransferase Dnmt3a: interaction with damaged DNA substratesтезисы доклада
- Авторы: Gromova Elizaveta S., Kirsanova Olga V., Sergeev Alexander S., Arutyunyan Alexander M.
- Сборник: Book of abstracts of International conference BIOCATALYSIS-2015: fundamentals&applications, June 21-26 2015, Moscow region, Russia
- Тезисы
- Год издания: 2015
- Первая страница: 44
- Последняя страница: 46
- Аннотация: 6-Thioguanine (SG) is anticancer agent but the mechanism of its therapeutic action is still not understood. The incorporation of SG into the CpG sequences instead of G could affect DNA methylation and, thus, represent a potential epigenetic mechanism of its cytotoxic effects. To elucidate the impact of SG lesions on DNA methylation, we prepared SG containing 30-mer DNA with one or two hemimethylated СpG sites (SG-DNA) and assessed their binding and methylation by catalytic domain of Dnmt3a (Dnmt3a-CD). The employment of the hemimethylated oligodeoxynucleotide duplexes as substrates allowed us to examine the effects of SG on methylation of a particular DNA strand in each CpG-site. Incorporation of SG into the substrate DNA differently affects methylation of CpG sites by Dnmt3a-CD. The influence strongly depends on the position of the modification.Binding of Dnmt3a-CD to Gs-DNA shows positive binding cooperativity like in the case of unmodified substrates. Introduction of the SG residue does not have a significant impact on the formation of the enzyme-substrate complex. The Kd values for Gs-DNA/ Dnmt3a-CD complexes slightly increased only in the case of incorporation of two SG residue in DNA. Absorption band of 6-thioguanine above 300 nm where unmodified DNA and the enzyme are transparent allowed us to use 6-thioguanine residue as spectroscopic probe to investigate whether 6-thioguanine incorporation into the CpG site affects flipping of the adjacent target cytosine by Dnmt3a-CD. The experiments with model prokaryotic MTase M.SssI showed that CD above 300 nm is sensitive to the local structural changes within the SG-DNA in SG-DNA/ M.SssI complexes. Addition of Dnmt3a-CD to the substrate with SG substitution 3’-adjacent to the target cytosine slightly changed the CD spectrum above 300 nm. The local disturbance of stacking interactions of bases in the CpG-site within SG-DNA/ Dnmt3a-CD complexes were observed probably due to the flipping of the target cytosine out of the double helix. According to the preliminary data a decrease in methylation of this substrate is not associated with worsening of the target cytosine flipping.
- Добавил в систему: Громова Елизавета Сергеевна