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Chernukhin V.A.,SibEnzyme Ltd. | Gonchar D.A.,SibEnzyme Ltd. | Kashirina Y.G.,SibEnzyme Ltd. | Degtyarev S.K.,SibEnzyme Ltd.
Biochemistry (Moscow) | Year: 2010

Optimal conditions for DNA methylation by the M3.BstF5I enzyme from Bacillus stearothermophilus and kinetic parameters of λ phage DNA modification and that of a number of oligonucleotide substrates are established. Comparison of M1.BstF5I and M3.BstF5I kinetic parameters revealed that with similar temperature optima and affinity for DNA, M3.BstF5I has nearly fourfold lower turnover number (0.24 min-1) and modifies the hemimethylated recognition site with lower efficiency under optimal conditions than the unmethylated one. In contrast to another three methylases of the BstF5I restriction-modification system, the M3.BstF5I enzyme is able to optionally modify the noncanonical 5′-GGATC-3′ DNA sequence with a rate more than one order of magnitude lower than the methylation rate of the canonical 5′-GGATG-3′ recognition site. © 2010 Pleiades Publishing, Ltd.

Abdurashitov M.A.,SibEnzyme Ltd. | Chernukhin V.A.,SibEnzyme Ltd. | Gonchar D.A.,SibEnzyme Ltd. | Dedkov V.S.,SibEnzyme Ltd. | And 2 more authors.
Research Journal of Pharmaceutical, Biological and Chemical Sciences | Year: 2016

Methyl-directed site-specific DNA endonuclease GlaI recognizes and cleaves methylated DNA sequence RCGY. GlaI activity in hydrolysis of recognition sequence containing two 5-methylcytosines is noticeably lower than in the case of the site with four or three 5-methylcytosines. We have shown that addition of DMSO to the reaction mixture significantly increases GlaI activity in hydrolysis of sites with two 5-methylcytosines but doesn't change a substrate specificity of the enzyme. A reaction buffer with DMSO may be used in epigenetic studies for GlaI digestion of eukaryotic genomes containing methylated CG pairs.

Dubinin E.V.,SibEnzyme Ltd. | Akishev A.G.,SibEnzyme Ltd. | Abdurashitov M.A.,SibEnzyme Ltd. | Oleynikova S.B.,SibEnzyme Ltd. | And 2 more authors.
Research Journal of Pharmaceutical, Biological and Chemical Sciences | Year: 2016

Real-time GlaI-PCR assay is developed to determine DNA methylation status of the regulation regions of HDAC4, URB1 and RARB genes in DNA preparations from human leukocytes. Real-time GlaI-PCR assay is DNA hydrolysis with methyl-directed site-specific DNA endonuclese GlaI followed by real-time PCR from the primers located upstream and downstream of the studied DNA region. The obtained data confirm a full methylation of the studied ACGT and GCGC sites in the regulatory regions of the HDAC4 and URB1 genes and a complete hydrolysis of these sites with GlaI. A first exon of RARB gene is slightly methylated in the leukocytes DNA preparations and according to the results of GlaI-PCR assay we don't observe GlaI hydrolysis of ACGCG site in RARB gene. The data obtained correspond to the literature data. The proposed method of real-time GlaI-PCR assay may be used to determine the methylation status of any unique parts in human and mammalian genomes.

Abdurashitov M.A.,SibEnzyme Ltd. | Gonchar D.A.,SibEnzyme Ltd. | Chernukhin V.A.,SibEnzyme Ltd. | Tomilov V.N.,SibEnzyme Ltd. | And 6 more authors.
BMC Genomics | Year: 2013

Background: Previously, we developed a simple method for carrying out a restriction enzyme analysis of eukaryotic DNA in silico, based on the known DNA sequences of the genomes. This method allows the user to calculate lengths of all DNA fragments that are formed after a whole genome is digested at the theoretical recognition sites of a given restriction enzyme. A comparison of the observed peaks in distribution diagrams with the results from DNA cleavage using several restriction enzymes performed in vitro have shown good correspondence between the theoretical and experimental data in several cases. Here, we applied this approach to the annotated genome of Drosophila virilis which is extremely rich in various repeats. Results: Here we explored the combined approach to perform the restriction analysis of D. virilis DNA. This approach enabled to reveal three abundant medium-sized tandem repeats within the D. virilis genome. While the 225 bp repeats were revealed previously in intergenic non-transcribed spacers between ribosomal genes of D. virilis, two other families comprised of 154 bp and 172 bp repeats were not described. Tandem Repeats Finder search demonstrated that 154 bp and 172 bp units are organized in multiple clusters in the genome of D. virilis. Characteristically, only 154 bp repeats derived from Helitron transposon are transcribed.Conclusion: Using in silico digestion in combination with conventional restriction analysis and sequencing of repeated DNA fragments enabled us to isolate and characterize three highly abundant families of medium-sized repeats present in the D. virilis genome. These repeats comprise a significant portion of the genome and may have important roles in genome function and structural integrity. Therefore, we demonstrated an approach which makes possible to investigate in detail the gross arrangement and expression of medium-sized repeats basing on sequencing data even in the case of incompletely assembled and/or annotated genomes. © 2013 Abdurashitov et al.; licensee BioMed Central Ltd.

Dedkov V.S.,SibEnzyme Ltd. | Gonchar D.A.,SibEnzyme Ltd. | Abdurashitov M.A.,SibEnzyme Ltd. | Udalyeva S.G.,SibEnzyme Ltd. | And 4 more authors.
Research Journal of Pharmaceutical, Biological and Chemical Sciences | Year: 2015

A fragment of Flavobacterium aquatile NL3 DNA carrying the gene of DNA methyltransferase M. FatI was cloned in pUC19 plasmid. DNA was sequenced and M. FatI gene was analyzed. A recombinant strain Esherichia coli was grown up and the enzyme was purified. M. FatI specificity was determined by a blocking of some restriction endonucleases and computer modeling. It's well known that M. NlaIII produces 5'-C(m6A)TG-3', whereas FatI MTase modifies the cytosine residue with formation 5'-(m5C)ATG-3'. The sensitivity of restriction endonucleases to FatI-methylation has been studied.

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