ИСТИНА

Methylation of CpG-sites in the DNA is an epigenetic modification of the genomes that plays an important role in regulation of many biologic processes including gene regulation. DNA methyltransferase (MTase) Dnmt3a is involved in the establishment in the genome de novo methylation pattern, the violation of which is associated with the development of malignant diseases. 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. According to X-ray analysis the active form of Dnmt3a-CD is a tetramer with two catalytic centers which could methylate opposite DNA strands of two CpG sites separated by 8–10 base pairs [1]. First, we have shown that methylation and binding of the unmodified substrates which contained two hemimethylated CpG sites with 5-methylcytosine residues either within the same or opposite DNA strands at a distance of 9 bp proceed with the equal efficiency. Probably only one active center of the enzyme is involved in the catalytic act. This information is important for establishing the correct pattern of methylation in cells. 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. The sensitivity of Dnmt3a to SG incorporation (reduction of methylation) is primarily associated with the modifications positioned within a CpG site 3’-adjacent to the target cytosine. This effect may be due to the perturbation of the contact of Dnmt3a-CD with the 6-oxo group of the guanine. It is consistent with our previous findings about importance of the contact of the 6-oxo group of the CpG guanine residue with Dnmt3a-CD [2]. Occurrence of the SG opposite the target cytosine or in the CpG flanking sequences virtually has no effect on the methylation efficiency. Importantly, that in the case of two-site substrates incorporation of one or two SG residues caused approximately equal decrease of the V0 values. Probably, methylation of both sites by two catalytic centers of tetramer Dnmt3a-CD occurs interdependently and modification of one of the CpG-sites affects methylation of the non-modified neighboring CpG site. 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. Another reason for the deterioration of the methylation may be a violation of necessary structural rearrangements of SG-DNA substrate. To address this issue, we have used the circular dichroism (CD) in the region of 300-400 nm. One of the main steps in the methylation process is flipping of the target cytosine out of the double helix for its methylation. 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. We showed previously that a number of guanine residue damages within the CpG sites such as 8-oxoguanine [3], O6-methylguanine [4], and adducts of guanine with benzo[a]pyrene diol epoxides [5] have a significant impact on the functioning of the Dnmt3a. Taken together, these results showed that 6-thioguanine, after being incorporated into DNA, as well as the other types of the guanine damages may perturb the epigenetic pathway of gene regulation.