| Recognition site | DNA sequence with at least three 5mC:
GCN↑GC
CG↓NCG
|
| Source | Planomicrobium koreense 78k |
| Substrate specificity | The enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA [1].
PkrI cleaves DNA sequence 5`- GCNGC-3`/3`-CGNCG-5', if at least three 5-methylcytosines are present in the recognition site (N isn`t considering).
Optimal recognition site (100% activity )
5`-G(5mC)NG(5mC)-3`/3`-(5mC)GN(5mC)G-5`
5`-G(5mC)NGC-3`/3`-(5mC)GN(5mC)G-5`
5`-GCNG(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 three sites:
5`-G(5mC)NG(5mC)-3`/3`-CGN(5mC)G-5` [2]. |
| Unit definition | One unit is defined as the amount of enzyme
required to hydrolyze completely 1 μg of linearized plasmid pFsp4HI3 in 1 hour at 37°C in a total reaction volume of 50 μl (see run 4 in the figure). |
PkrI activity assay on DNA pFsp4HI3/DriI
Lanes:
1 and 6 – 1 Kb SE DNA-markers.
2 – Control DNA pFsp4HI3/DriI,
3 – 0.5 μl Pkr I
4 – 1 μl Pkr I
5 – 2 μl Pkr I
Products were separated in 1,4% agarose gel in Buffer TAE. |
 . |
| Reaction buffer | SE-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 | 37oC |
| Storage conditions | 20 mM Tris-HCl (pH 7.4); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 50% glycerol; Store at -20°C. |
| Non-specific hydrolisis | No detectable degradation of 1μg of Lambda DNA was observed after incubation with 2 units of enzyme for 16 hours at 37°C in a total reaction volume of 50 μl.
|
| Reagents Supplied with Enzyme |
10 X SE-buffer Y |
| 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, T. N. Nayakshina, D.A. Gonchar, J.E. Tomilova, M.V. Tarasova, V.S. Dedkov, N.A. Mikhnenkova, S.Kh. Degtyarev A new site-specific methyl-directed DNA endonuclease pkri recognizes and cuts methylated DNA sequence 5'-GCN^GC-3'/3'-CG^NCG-5' carrying at least three 5-methylcytosines // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.7, No 3, pp 35-42, 2011
2. Chmuzh E.V., Kashirina Yu.G., Tomilova Yu.E., Chernukhin V.A., Okhapkina S.S., Gonchar D. A., Dedkov V.S., Abdurashitov M. A., Degtyarev S. Kh. Gene cloning, comparative analysis of the protein structures from Fsp4HI restriction-modification system and biochemical characterization of the recombinant DNA methyltransferase M.Fsp4HI. // Molecular Biology, V.41, No 1, p. 43-50 (2007)
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