Product info: Pkr I


Name
Pkr I
Cat. #E579E580
Package, u.a.50250
Concentration, u.a./ml10001000

Recognition site
DNA sequence with at least three 5mC:
GCN↑GC
CG↓NCG
SourcePlanomicrobium koreense 78k
Substrate specificityThe 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 onDNA 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 definitionOne 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.
PkrI activity assay on DNA pFsp4HI3/DriI
.
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
50 - 7575 - 10010 - 2525 - 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
37oC
Storage conditions20 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 hydrolisisNo 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 sensitivityThe 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)