Product info: Gla I


Name
Gla IDoes not cleave DNA sequence G(4mC)G(4mC)  
Cat. #E493E494
Package, u.a.100500
Concentration, u.a./ml1000010000

Recognition site
Pu(5mC)↑GPy
PyG↓(5mC)Pu
SourceGlacial ice bacterium GL29
Substrate specificityThe enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA and DNA with N4-methylcytosines [1].
The enzyme activity depends on number and position of methylated nucleotides in the recognition sequence [4]:
Optimal substrate (100% activity )
5`-G(5mC)G(mC)-3`/3`-(5mC)G(5mC)G-5`
Good substrates ( > 25% activity)
5`-R(5mC)G(5mC)-3`/3`-YG(5mC)G-5`
5`-A(5mC)GT-3`/3`-TG(5mC)A-5`
Medium substrates ( > 6% activity)
5`-G(5mC)R(5mC)-3`/3`-(5mC)GYG-5`
5`-G(5mC)GT-3`/3`-CG(5mC)A-5`
Bad substrates (6% activity)
5`-G(5mC)GC-3`/3`-CG(5mC)G-5`
Assayed onDNA pHspAI2/GsaI is a linearized plasmid pHspAI2, which carries a gene of DNA-methyltransferase M.HspAI (recognition sequence 5`-GCGC-3`) and includes a unique GlaI recognition site
5`-G(5mC)G(5mC)-3`/3`-(5mC)G(5mC)G-5` [2].
Unit definitionOne unit is defined as the amount of enzyme required to completely digest a unique 5`-G(5mC)G(5mC)-3`/3`-(5mC)G(5mC)G-5` site in 1 μg of pHspAI2 plasmid DNA, which is linearized with GsaI, in 1 hour at 30°C in a total reaction volume of 50 μl. As a result of this site hydrolysis two main DNA fragments (3,419 and 699 bp) are produced (see lanes 3-5 in the figure). GlaI digestion of recognition sequences with three and two 5-methylcytosines results in additional bands appearance (lane 6 in the figure).
GlaI activity assay on DNA pHspAI2/GsaI

Lanes:
2 Control pHspAI2/GsaI DNA
3 - 0.5 μl GlaI (diluted 1/20),
4 - 1 μl GlaI (diluted 1/20),
5 - 2 μl GlaI (diluted 1/20),
6 - 1 μl of undiluted GlaI,
1 and 7- 1 Kb SE DNA Ladder.

Products were separated in 1% agarose gel in TAE Buffer.
GlaI activity assay on DNA pHspAI2/GsaI
Optimal SE-bufferY (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.)
Enzyme activity (%)
BGOWYRose
75 - 10075 - 10025 - 5025 - 50100100
Reaction bufferSE-buffer Y, (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.)
Optimal temperature
30oC
Storage conditions10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 0,05% Triton X-100, 0.1 mg/ml BSA, 50% glycerol; Store at -20C.
Non-specific hydrolisisNo detectable degradation of 1μg of Lambda DNA was observed after incubation with 8 units of enzyme for 16 hours at 30C in a total reaction volume of 50 μl.
Reagents Supplied with Enzyme 10 X SE-buffer Y, pHspAI2/GsaI DNA
Methylation sensitivityThe enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA and DNA with N4-methylcytosines [1].
Inactivation 20 minutes under
65oC
MSDS:Download MSDS as PDF
References:1. Chernukhin V.A., Nayakshina T.N., Tomilova J.E., Mezentseva N.V., Dedkov V.S., Degtyarev S.Kh. Bacterial strain Glacial ice bacterium I - producer of GlaI restriction endonuclease. // Russian Federation patent RU 2287012 C1 (2006).
2.
 Valery A. Chernukhin, Tatyana N. Najakshina, Murat A. Abdurashitov, Julia E. Tomilova, Nina V. Mezentzeva, Vladimir S. Dedkov, Natalya A. Mikhnenkova, Danila A. Gonchar, Sergei Kh. Degtyarev A novel restriction endonuclease GlaI recognizes methylated sequence 5-G(m5C)^GC-3 // Biotechnologia (russ.). 2006. N 4. P. 31-35
3.  Tomilova J.E., Chernukhin V.A., Degtyarev S.Kh. Dependence of site-specific endonuclease GlaI activity on quantity and location of methylcytosines in the recognition sequence 5-GCGC-3. // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.2, No 1, pp 30-39 (2006) (Russian)
4.  G. V. Tarasova, T. N. Nayakshina, S. Kh. Degtyarev Substrate specificity of new methyl-directed DNA endonuclease GlaI // BMC Molecular Biology 2008, 9:7
5. Zemlyanskaya, E.V., Degtyarev, S.K. Substrate specificity and properties of methyl-directed site-specific DNA endonucleases.// Molecular Biology, v.47, No.6, p.900-913 (2013)

Application:
 Chernukhin V.A, Abdurashitov M.A., Tomilov V.N., Gonchar D.A., Degtyarev S.Kh. Comparative analysis of mouse chromosomal DNA digestion with restriction endonucleases in vitro and in silico // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.3, No 4, pp 19-27, 2007
Abdurashitov M.A., Chernukhin V.A, Gonchar D.A., Degtyarev S.Kh. GlaI digestion of mouse γ-satellite DNA: study of primary structure and ACGT sites methylation // BMC Genomics 2009, 10:322.
 D. A. Gonchar, A. G. Akishev, S. Kh. Degtyarev BlsI- and GlaI-PCR assays a new method of DNA methylation study // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.6, No 1, pp 5-12, 2010
Wood, R. J., McKelvie, J. C., Maynard-Smith, M. D., and Roach, P. L. A real-time assay for CpG-specific cytosine-C5 methyltransferase activity.// (2010) Nucleic Acids Res 38, e107
Kravets AP, Mousseau TA, Litvinchuk AV, Ostermiller Sh, Vengen GS, Grodzinski DM. Changes in wheat DNA methylation pattern after chronic seed gamma-irradiation.// Tsitol Genet. 2010 Sep-Oct;44(5):18-22. Russian.
 Alexander G. Akishev, Danila A. Gonchar, Murat A. Abdurashitov and Sergey Kh. Degtyarev Epigenetic typing of human cancer cell lines by BlsI- and GlaI-PCR assays // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.7, No 3, pp 5-16, 2011
F. Syeda, R.L. Fagan, M. Wean, G.V. Awakumov, J.R. Walker, S. Xue, S. Dhe-Paganon, & C. Brenner, "The RFTS Domain is a DNA-competitive Inhibitor of Dnmt1"//, JBC, v. 286, pp. 15344-15351 (2011).
Keith N. Rand, Graeme P. Young, Thu Ho and Peter L. Molloy , "Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylationdependent restriction enzymes."//, Nucleic Acids Research, pp. 1-10 (2012).
Fagan RL, Wu M, Chedin F, Brenner C An Ultrasensitive High Throughput Screen for DNA Methyltransferase 1-Targeted Molecular Probes //PLoS ONE 8(11): e78752. doi:10.1371/journal.pone.0078752 (2013)
Kuznetsov V.V., Abdurashitov M.A., Akishev A.G., Degtyarev S.H. Method for detection nucleotide sequence R(5mC)GY in given position of continuous DNA.// Russian Federation patent RU 2525710 C1 (2014).