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In mammals, de novo methylation of cytosine residues in DNA CpG sites is catalyzed by DNA methyltransferase (MTase) Dnmt3a. Changes in the CpG methylation status in gene promoters are crucial for the regulation of their expression. Some of these CpGs are located adjacent to G-quadruplex (G4) structures that can potentially affect the activity of MTases. Here, we aimed to investigate the relationship between the presence of parallel G4s in DNA and the functioning of the Dnmt3a in vitro using model DNAs based on proto-oncogene promoters. Fluorescence polarization measurements revealed that Dnmt3a catalytic domain (Dnmt3a-CD), in contrast to the regulatory PWWP domain, binds to G4 oligonucleotide of a sequence based on the c-MYC promoter. The activity of Dnmt3a-CD was strongly inhibited in the presence of the G4 oligonucleotide. Moreover, the extent of methylation of short G4-containing c-MYC DNA duplexes was reduced >3-fold compared to control DNA substrates. Using a novel nanopore sequencing method, we analyzed the in vitro methylation of the model 752-bp proto-oncogene promoter region containing 94 CpG sites as well as G4s. Unexpectedly, we discovered a predominantly methylated region coinciding with G4-forming sequences of the promoter. These effects may be explained by Dnmt3a-CD sequestration at G4 structures, resulting in an effective targeting of nearby CpG sites by the enzyme. However, a rigid G4 structure, such as in the case of short c-MYC G4 DNA duplexes and oligonucleotides, may hinder the efficient functioning of Dnmt3a-CD. The research was supported by RSF (project No. 22-24-00368) and MSU Program of Development (project No. 23-Sh04-45)
№ | Имя | Описание | Имя файла | Размер | Добавлен |
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1. | Программа конференции | 1st_ES_Meeting_-_Final_program.pdf | 10,3 МБ | 20 декабря 2023 [AVSergeev1992] | |
2. | Полный текст | Тезисы | 2023Epigenetic_Soc_Abstract_Book.pdf | 442,9 КБ | 23 ноября 2023 [AVSergeev1992] |