Peculiarities of the Regulation of Gene Expression in the Ecl18kI Restriction-Modification System.

Transcription regulation in bacterial restriction-modification (R-M) systems is an important process, which provides coordinated expression levels of tandem enzymes, DNA methyltransferase (MTase) and restriction endonuclease (RE) protecting cells against penetration of alien DNA. The present study focuses on (cytosine-5)-DNA methyltransferase Ecl18kI (M.Ecl18kI), which is almost identical to DNA methyltransferase SsoII (M.SsoII) in terms of its structure and properties. Each of these enzymes inhibits expression of the intrinsic gene and activates expression of the corresponding RE gene via binding to the regulatory site in the promoter region of these genes. In the present work, complex formation of M.Ecl18kI and RNA polymerase from Escherichia сoli with the promoter regions of the MTase and RE genes is studied. The mechanism of regulation of gene expression in the Ecl18kI R-M system is thoroughly investigated. M.Ecl18kI and RNA polymerase are shown to compete for binding to the promoter region. However, no direct contacts between M.Ecl18kI and RNA polymerase are detected. The properties of M.Ecl18kI and M.SsoII mutants are studied. Amino acid substitutions in the N-terminal region of M.Ecl18kI, which performs the regulatory function, are shown to influence not only M.Ecl18kI capability to interact with the regulatory site and to act as a transcription factor, but also its ability to bind and methylate the substrate DNA. The loss of methylation activity does not prevent MTase from performing its regulatory function and even increases its affinity to the regulatory site. However, the presence of the domain responsible for methylation in the M.Ecl18kI molecule is necessary for M.Ecl18kI to perform its regulatory function.

SsoII-like DNA-methyltransferase Ecl18kI: interaction between regulatory and methylating functions.

The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Delta(72-379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyltransferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.

[Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues].

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue into the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of the unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

[Study of heterogeneity of chromatin endonuclease fragments, using fractionation by step-wise endonucleolysis]. / Issledovanie geterogennosti endonukleaznykh fragmentov khromatina. Fraktsionirovanie metodom stupenchatogo endonukleoliza.

Isolated nuclei from rat liver were incubated at different time intervals under conditions, optimal for manifestation of the Ca, Mg-dependent endonuclease activity. After each of the 6 endonucleolyses chromatin was extracted and 6 chromatin fractions (I--VI) and "residual" chromatin were collected. A comparative analysis of the "early" (I--III) and "late" chromatin fractions revealed an increased RNA content in the "late" fractions and changes in the composition of the non-histone proteins. Electrophoresis in acrylamide gel concentration gradient demonstrated that fractions I--III predominantly contain high molecular weight fragments whereas fractions IV-VI are represented by highly fragmented chromatin. Each fraction was characterized by peculiar shapes of alkaline denaturation curves and by heterogeneity of charges of their constituent chromatin fragments.

[Study of heterogeneity of the chromatin endonuclease fragment by free flow electrophoresis]. / Issledovanie geterogennosti endonukleaznykh fragmentov khromatina elektroforezom v svobodnom potoke.

The oligomer chromatin fragments relatively uniform in size (8--11 nucleosomes) were prepared by a short-term endonycleolysis. The heterogeneity of these fragments with respect to their electrophoretic mobility was revealed using free flow electrophoresis. The individual fragmentated chromatin subfractions were obtained. These subfractions differed in their protein and RNA content per DNA weight unit, in quantitative ratios of different zones of high molecular weight non-histone proteins and in thermal and alkaline denaturation kinetics. It was also found that the parameters investigated are correlated with electronegativeity of the fragmentated chromatin subfractions.