| Recognition site | G(5mC)↑NG(5mC)
(5mC)GN↓(5mC)G |
| Source | Glacial ice bacterium GL24 |
| Substrate specificity | The enzyme cleaves C5-methylated DNA
and does not cut unmodified DNA [1].
Optimal recognition site (100% activity ):
5'-G(5mC)↑NG(5mC)-3'/3'-(5mC)GN↓(5mC)G-5` |
| Assayed on | DNA pFsp4HI3/DriI is a linearized plasmid pFsp4HI3, which carries a gene of DNA-methyltransferase M.Fsp4HI and includes a unique GluI site:
5`-G(5mC)NG(5mC)-3`/3`-(5mC)GN(5mC)G-5` [2]. |
| Unit definition | One unit is defined as the amount of enzyme
required to hydrolyze completely a unique site:
5`-G(5mC)NG(5mC)-3`/3`-(5mC)GN(5mC)G-5`
in 1 μg of linearized plasmid pFsp4HI3/DriI in 1 hour at 50°C in a total reaction volume of 50 μl. As a result a linearized plasmid pFsp4HI3 disappears (see run 4 in the figure). GluI digestion of recognition sequences with three 5-methylcytosines results in additional bands appearance (runs 4-6 in the figure).
|
GluI activity assay on DNA pFsp4HI3/DriI
Lanes:
1 and 6 – 1 Kb SE DNA-markers.
2 – Control DNA pFsp4HI3/DriI,
3 – 0.5 μl Glu I
4 – 1 μl Glu I
5 – 2 μl Glu I
Products were separated in 1,4% agarose gel in Buffer TAE. |
|
| Reaction buffer | SE-buffer 2K, (10 mM Tris-HCl (pH 7.6 at 25°C); 10 mM MgCl2; 200 mM KCl; 1 mM DTT.) |
| Optimal temperature | 50oC |
| Storage conditions | 10 mM Tris-HCl (pH 7.5); 200 mM KCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 200 μg/ml BSA, 50% glycerol; Store at -20°C. |
| Non-specific hydrolisis | No detectable degradation of 1μg of Lambda DNA was observed after incubation with 1 units of enzyme for 16 hours at 50°C in a total reaction volume of 50 μl.
|
| Reagents Supplied with Enzyme |
10 X SE-buffer 2K |
| Methylation sensitivity | The enzyme cleaves C5-methylated DNA
and does not cut unmodified DNA [1].
|
| Inactivation 20 minutes under | 65oC |
| Notes | When using a buffer other than the optimal (suppied) SEBuffer, it may be necessary to add more enzyme to achieve complete digestion. |
| MSDS: | Download MSDS as PDF |
| References: | 1. Chernukhin V.A., Chmuzh E.V., Tomilova J.E., Nayakshina T.N., Dedkov V.S., Degtyarev S.Kh. Bacterial strain Glacial ice bacterium - producer of GluI site specific endonuclease. // Russian Federation patent RU 2322492 C1 (2006).
2.  V.A. Chernukhin, E.V. Chmuzh, Yu.E. Tomilova, T.N. Nayakshina, D.A. Gonchar, V.S. Dedkov, S.Kh. Degtyarev A novel site-specific endonuclease GluI recognizes methylated DNA sequence 5’-G(5mC)^NG(5mC)-3’/3’-(5mC)GN^(5mC)G. // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.3, No 2, pp 13-17, 2007
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
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.
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