| Recognition site | (5mC)GNNNNN↑NN(5mC)G
G(5mC)NN↓NNNNNG(5mC)
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| Source | Paracoccus carotinifaciens 3K |
| Substrate specificity | The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA [1].
The enzyme activity depends on nucleotides neighboring to a recognition site and a number of methylated cytosines.
Optimal recognition site (100% activity ):
5'-W(5mC)GNNNNNNN(5mC)GW-3'/
3'-WG(5mC)NNNNNNNG(5mC)W-5` |
| Assayed on | pMHgaI/DriI is a linearized plasmid pMHgaI, which carries genes of DNA-methyltransferases M1.HgaI (recognition sequence 5`-GCGTC-3`) and M2.HgaI (5`-GACGC-3`) and includes a unique PcsI canonical site:
5`-W(5mC)GNNNNNNN(5mC)GW-3`/
3`-WG(5mC)NNNNNNNG(5mC)W-5` [1]. |
| Unit definition | One unit is defined as the amount of enzyme required to digest a unique site
5`-A(5mC)GNNNNNNN(5mC)GT-3`
in 1 μg of DNA pMHgaI/DriI in 1 hour at 37°C in a total reaction volume of 50 μl.
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PcsI activity assay on DNA pMHgaI/DriI
Lanes:
1 and 6 – 1 Kb SE DNA-markers
2 – Control pMHgaI/DriI
3 – 0.5 μl Pcs I
4 – 1 μl Pcs I
5 – 2 μl Pcs I
Products were separated in 1,4% agarose gel in Buffer TAE.
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| Optimal SE-buffer | SE-buffer PcsI (10 mM Tris-HCl (pH 8.3 at 25°C); 20mM NaCl, 3 mM MgCl2, 1 mM DTT.) |
| Enzyme activity (%) | | B | G | O | W | Y | Rose | | 50 - 75 | 25 - 50 | 0 - 10 | 10 - 25 | 50 - 75 | 20 |
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| Reaction buffer | SE-buffer PcsI, (10 mM Tris-HCl (pH 8.3 at 25°C); 20mM NaCl, 3 mM MgCl2, 1 mM DTT.) |
| Optimal temperature | 37oC |
| Storage conditions | 10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 0.1 mg/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 37°C in a total reaction volume of 50 μl.
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| Reagents Supplied with Enzyme |
10 X SE-buffer PcsI |
| Methylation sensitivity | The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA [1].
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| 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., Nayakshina T.N., Tarasova M.V., Golikova L.N., Akishev A.G., Dedkov V.S., Mikhnenkova N.A., Degtyarev S.Kh. Bacterial strain Paracoccus carotinifaciens 3K- producer of PcsI site specific endonuclease. // Russian Federation patent RU 2377294 C1 (2009).
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