We have identified a bacterial strain, Peribacillus species 31, which produces a novel restriction endonuclease named Pse31I. The enzyme recognizes the six-nucleotide non-palindromic DNA sequence 5’-GGTCTC-3’ and cleaves it downstream of the recognition site at positions 1/5: 5’-GGTCTC(N)1^/3’-CCAGAG(N)5^-5’. Thus, Pse31I is a true isozomere of the Eco31I restriction enzyme. The producing strain was identified based on morphological and biochemical characteristics, as well as on analysis of the primary structure of a fragment of the 16S rRNA gene. Pse31I exhibits maximum activity at 37°C and is inactivated by incubation at 55 and 65°C. The enzyme does not cleave sites containing methylated cytosine in the upper strand (5’-GGTCT(5mC)-3’) and/or in the lower strand (3’-(5mC)CAGAG) .

The optimal conditions for enzyme activity are SE Buffer 5 (Y) (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and a temperature of 37°C.

A PecI restriction endonuclease preparation with a concentration of 10,000 units/mL was obtained through purification in four chromatographic stages. The optimal conditions for the enzyme reaction are SE Buffer 5 (Y) (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and a temperature of 37°C.

We have identified a bacterial strain, Brevundimonas mongoliensis 53, which produces a novel large-cleavage restriction endonuclease named BmoI. The BmoI enzyme recognizes the seven-nucleotide palindromic DNA sequence 5’-GG↓GWCCC-3’ (W = A or T) and cleaves it as indicated by the arrow, forming three-nucleotide
5’-overhanging “sticky” ends. BmoI is an isozomere of the previously described restriction endonuclease SanDI. The producer strain was identified based on morphological and biochemical characteristics, as well as on analysis of the primary structure of a fragment of the 16S rRNA gene. A preparation of the BmoI restriction endonuclease with a concentration of 2,000 units/mL was obtained by purification through three chromatographic stages. The optimal conditions for enzyme activity are SE buffer W (10 mM Tris-HCl (pH 8.5), 10 mM MgCl₂, 100 mM NaCl, 1 mM DTT) and a reaction temperature of 37°C.

We have identified a bacterial strain, Exiguobacterium mexicanum 6, which produces a novel restriction endonuclease named EmiS6I. The EmiS6I enzyme recognizes the six-nucleotide non-palindromic DNA sequence 5’-CTCTTC-3’ and cleaves it downstream of the recognition site at positions 1/4: 5’-CTCTTC(N)1↓-3’/3’-GAGAAG(N)4↓-5’, forming 5’-protruding trinucleotide ends. Thus, EmiS6I is an isozysomer of the restriction enzymes Ksp632I and Bst6I. The producer strain was identified based on morphological and biochemical characteristics, as well as on analysis of the primary structure of a fragment of the 16S rRNA gene. A preparation of the EmiS6I restriction endonuclease with a concentration of 5,000 units/mL was obtained by purification through three chromatographic steps. The optimal conditions for enzyme activity are SE Buffer Y (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and a temperature of 37°C.

The 5-methylcytosine-dependent DNA endonuclease GlaI recognizes and cleaves 5′-R(5mC)GY-3′ / 3′-YG(5mC)R-5′ sites in DNA, which are formed de novo in the human genome by the action of the DNA methyltransferase DNMT3. Due to its specificity, the GlaI enzyme is effectively used to determine the methylation status of RCGY sites in the human genome. Genomic mapping of R(5mC)GY sites is performed using next-generation sequencing (NGS) of DNA hydrolysates obtained by GlaI digestion, whereas analysis of individual DNA fragments can be performed using GlaI-PCR.

In this study, we compared the results of NGS analysis of GlaI-digested DNA hydrolysates with those of GlaI-PCR analysis of the regulatory regions of 11 tumor-suppressor genes in the DNA of the human cell lines L68 (normal lung fibroblasts), Raji (Burkitt’s lymphoma), and U937 (histiocytic lymphoma). It was shown that in L68 cell DNA, most of the regulatory regions studied are unmethylated, whereas in the DNA of the malignant cell lines Raji and U937, methylation of these regions is observed. With the exception of one region, for all other fragments studied, the methylation status determined by GlaI-PCR analysis fully correlates with the number of R(5mC)GY sites identified by NGS. The only discrepancy between the data from the two methods was identified in the regulatory region of the RASSF1A gene in L68 cell DNA, which is explained by the presence of allele-specific methylation, confirmed by an additional real-time GlaI-PCR analysis. Given the presence of allelic methylation in the regulatory region of the RASSF1A gene, we can conclude that there is complete agreement between the NGS data for GlaI hydrolysates and the GlaI-PCR analysis of the regulatory regions of the studied tumor suppressor genes in the human cell lines L68, Raji, and U937.

Thus, NGS analysis of GlaI-hydrolyzed DNA and GlaI-PCR analysis are complementary methods that enable the reliable identification of abnormally methylated genomic regions in normal and tumor cells, making them promising tools for epigenetic research and DNA diagnostics.

We have identified a bacterial strain, Paracoccus species 12, that produces a novel restriction endonuclease named PecI. This enzyme recognizes the six-nucleotide palindromic DNA sequence 5’-TTA^TAA-3’ and cleaves it to form “blunt” ends, as indicated by the arrow. Thus, PecI is a true isozym of the PsiI restriction endonuclease.

A PecI restriction endonuclease preparation with a concentration of 10,000 units/mL was obtained through a four-step chromatographic purification process. The optimal reaction conditions for the enzyme are SE Buffer 5 (Y) (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and a temperature of 37°C.