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6-Thioguanine (Gs) is a cytotoxic drug which is widely used for the treatment of acute leukemia. However, the mechanism of Gs cytotoxicity is still unknown. In vivo Gs forms 2’-deoxy-6-thioguanosine triphosphate which incorporates into DNA. DNA methylation at CpG sites is an epigenetic modification of the genomes that plays an important role in regulation of many biologic processes including gene regulation. DNA methyltransferase Dnmt3a is involved in establishment of new methylation patterns in genome. The incorporation of Gs into CpG sites may affect the activity of Dnmt3a. Here, we examine the impact of Gs on cytosine methylation mediated by catalytic domain of Dnmt3a (Dnmt3a-CD). 30-mer DNA containing Gs in one or two CpG sites separated by 10 base pairs (one helix turn) and in the flanking sequences (Gs-DNA) were obtained. an introduction of the Gs into CpG site near the target cytosine led to a 4 times decrease of methylation as compared to the unmodified substrate. It is consistent with our previous findings about crucial importance of the contact of the 6-oxo group of the CpG guanine residue with Dnmt3a-CD. Occurrence of the Gs opposite the target cytosine or in the CpG flanking sequence virtually has no effect on the efficiency of methylation. Incorporation of one or two Gs residues (in the case of two-site substrate) caused approximately equal decrease of the V0 values. Probably methylation of both sites by two catalytic centers of tetrameric Dnmt3a-CD occurs interdependently. The Kd values for Gs-DNA/ Dnmt3a-CD complexes slightly increased regardless of the position of the Gs residue in DNA. Taken together, these results showed that 6-thioguanine, after being incorporated into DNA, may perturb the epigenetic pathway of gene regulation.