ИСТИНА

The aim of this work was to study the methylation of nucleosomes as structural units of chromatin by full-size Dnmt3a and to construct a nucleosome containing B[a]P-damaged DNA for subsequent examination of its interaction with Dnmt3a. Highly purified full-size Dnmt3a was isolated by metal-affinity and cation exchange chromatography. Two types of 145-mer B[a]P-DNA were obtained. B[a]P residue covalently bound to the guanine residue of the CpG site was either in the DNA minor groove ((-)-trans-B[a]P-N2-dG) or intercalated into DNA ((+)-cis-B[a]P-N2-dG) with displacement of the modified guanine and partner cytosine out of the double helix structure. The upper strands of DNA duplexes with modified or unmodified central CpG site were obtained by ligation of 68-, 12- and 65-mer oligonucleotides on 145-mer template. B[a]P-DNA was obtained by annealing the upper strand, containing B[a]P-N2-dG adduct at the central 12-mer fragment, with the lower strand obtained by asymmetric PCR. Unmodified duplex was obtained by PCR. Reconstitution of these DNA duplexes with recombinant histones was performed by stepwise dialysis with the excess of the histones and in the absence of the competitive DNA yielding nucleosomes with unmodified DNA or B[a]P-DNA. Further methylation by Dnmt3a of 30-mer DNA duplex containing the central fragment with B[a]P-adduct within the CpG-site similar to that of 145-mer was studied. Introduction of minor groove and intercalative B[a]P adducts led to the 30- and 13-fold diminution of DNA methylation rate correspondingly. Dnmt3a interaction with nucleosomes demonstrated that nucleosome methylation was impaired 9-27 times compared to free DNA. The degree of nucleosome methylation did not change after the enzyme:substrate ratio achieved 4:1 in accordance with tetrameric Dnmt3a structure. It is necessary to note that Dnmt3a reacts with nucleosome more efficiently than its catalytic domain presumably due to its interaction with histone H3.