Murat A. Abdurashitov, Valery A. Chernukhin, Danila A. Gonchar, Vladimir S. Dedkov, Natalia A. Mikhnenkova, and Sergey Kh. Degtyarev
Research Journal of Pharmaceutical, Biological and Chemical Sciences, Volume 7, Issue 1, 2016 (January - February)
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.
VA Chernukhin, DA Gonchar, MA Abdurashitov, EV Kileva, OA Belichenko, VS Dedkov, NA Mikhnenkova, SKh Degtyarev
RJPBCS, vol 7(2), 2016, p.p. 388-394
A comparative analysis of biochemical properties of three similar site specific methyl-directed DNA endonucleases BisI, BlsI and EcoBLI recognizing 5’-G(5mC)NGC-3’/3’-CGN(5mC)G-5’ has been done. Optimal concentrations of MgCl2, NaCl or KCl, pH of reaction buffers as well as the optimal reaction temperatures for these enzymes were determined. It was shown that BisI has the maximal activity in the buffer 10 mM Tris-HCl (pH 9.0 at 250 C), 15 mM MgCl2, 150 mM KCl, 1 mM DTT. An optimal buffer for MD-endonuclease BlsI is 10 mM Tris-HCl (pH 8.0 at 250 C), 15 mM MgCl2, 100 mM NaCl, 1 mM DTT. The enzyme EcoBLI displays a maximal activity in the buffer 10 mM Tris-HCl (pH 8.5 at 250 C), 10 mM MgCl2, 25 mM NaCl, 1 mM DTT. Optimal temperature for all enzymes is 370 C
BMC Molecular Biology 2008, 9:7
Recently, we have discovered site-specific endonucleases, which recognize and cleave only DNA sequences with 5-methylcytosine. Two specificities of such endonucleases have been described. Enzymes BisI, BlsI, and GluI are isoschizomers and hydrolyze the DNA sequence 5'-GCNGC-3'/3'-CGNCG-5', which is methylated in different ways. The enzyme GlaI cleaves the DNA sequence 5'-GCGC-3'/3'-CGCG-5' if there are two, three or four 5-methylcytosines. The goal of the present work is to study in detail the composition of recognition sequence and effect of the methylated cytosines on the efficiency of DNA cleavage by the methyl-directed DNA endonuclease GlaI. In a recent work we have studied the dependence of GlaI activity on the quantity and location of 5-methylcytosines in the enzyme recognition sequence 5'-GCGC-3'/3'-CGCG-5'. A significant DNA cleavage has been observed for oligonucleotide duplexes, which include either three or four 5-methylcytosines. In this work we have studied dependence of the GlaI activity on quantity and location of methylated cytosines, as well as on composition of the recognition sequence.The list of good substrates for GlaI includes a fully methylated site 5'-CGCG-3'/3'-GCGC-5', sites with three cytosines of a general structure 5'-PuMGM-3'/3'-PyGMG-5', and one recognition sequence with two methylated cytosines 5'-AMGT-3'/3'-TGMA-5', where M is 5-methylcytosine. GlaI intermediate substrates include sites with three methylated cytosines of a general structure 5'-GMPuM-3'/3'-MGPyG-5', as well as a site with two methylcytosines 5'-GMGT-3'/3'-CGMA-5'. The site 5'-GMGC-3'/3'-CGMG-5' may be considered a low activity substrate.
Translated from "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.2, No 1, pp 30-39 (2006)
The activity dependence of site-specific endonuclease GlaI that recognizes and hydrolyzes only methylated DNA sequence 5’-GCGC-3’ on the quantity and location of 5-methylcytosines in enzyme’s recognition sequence has been studied. A significant DNA cleavage has been observed for oligonucleotides duplexes containing four and three 5-methylcytosines or two internal modified bases. The cleavage efficiency is maximal for DNA duplex with four 5-methylcytosine and decreases when a number of methylated bases are lower. GlaI hydrolyzes recognition sequences with 5-methylcytosines but not with N4-methylcytosines.