| Recognition site | G↓C(5mC)GGC
CGG(5mC)C↑G |
| Source | Kocurea rosea 307 |
| Substrate specificity | The enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA [1].
Kro I doesn`t cleave DNA modified with MspI DNA-methyltransferase
Recognition site
G↓C(5mC)GGC /
CGG(5mC)C↑G.
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| Assayed on | DNA pMHpaII 1/DriI is a linearized plasmid pMHpaII 1.
pMHpaII 1 carries a gene of DNA-methyltransferase M.HpaII, which methylates sites 5`-CCGG-3` producing 5`-C(5mC)GG-3`/3`-GG(5mC)C-5`, and includes three canonical sites
5`-GC(5mC)GGC-3`/3`-CGG(5mC)CG-5`. |
| Unit definition | One unit is defined as the amount of enzyme required to hydrolyze completely 1 μg of linearized plasmid pMHpaII 1 in 1 hour at 37°C in a total reaction volume of 50 μl.
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KroI activity assay on DNA pMHpaII 1/DriI
Lanes:
1 and 6 – 1 Kb SE DNA-markers.
2 – Control DNA pMHpaII 1/DriI,
3 – 0.5 μl Kro I
4 – 1 μl Kro I
5 – 2 μl Kro I
Products were separated in 1,4% agarose gel in Buffer TAE. |
 |
| Optimal SE-buffer | G (10 mM Tris-HCl (pH 7.6 at 25°C); 10 mM MgCl2; 50 mM NaCl; 1 mM DTT.) |
| Enzyme activity (%) | | B | G | O | W | Y | Rose | | 50 - 75 | 100 | 25 - 50 | 50 - 75 | 75 - 100 | 100 |
|
| Reaction buffer | SE-buffer G |
| Optimal temperature | 37oC |
| Storage conditions | 10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 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 30°C in a total reaction volume of 50 μl.
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| Reagents Supplied with Enzyme |
10 X SE-buffer G |
| Methylation sensitivity | The enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA [1]. |
| Inactivation 20 minutes under | 65oC |
| MSDS: | Download MSDS as PDF |
| References: | 1.  V.A. Chernukhin, E.V. Kileva, J.E. Tomilova, A.A. Boltengagen, V.S. Dedkov, N.A. Mikhnenkova, D.A. Gonchar, L.N. Golikova, S.Kh. Degtyarev A new methyl-directed site-specific endonuclease KroI recognizes and cuts DNA sequence 5’-G^C(5mC)GGC-3’ // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.7, No 1, pp 14-20, 2011
2. Chernukhin V.A., Zhuravleva R.O., Tarasova G.V., Boltengagen A. A., Akishev A.G., Mikhnenkova N.A., Degtyarev S.Kh. Bacterial strain Kocuria rosea - producer of KroI site specific endonuclease. // Russian Federation patent RU 2394099 C1 (2010).
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