| Recognition site | (5mC)GNNNNN↑NN(5mC)G
G(5mC)NN↓NNNNNG(5mC)
|
| Source | AnE.coli strain that carries the cloned P cs I
gene fromParacoccus carotinifaciens3K |
| 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 | pMHpySE49 plasmid DNA, linearized with Dr iI
(pMHpySE49/DriI). pMHpySE49 (3536 bp) was obtained by a ligation of the
vector pMTL22/FauNDI/SmaI and a PCR-fragment
containing M.HpySE526I (f orms 5'-A(5mC)GT-3') gene and
DNA sequence:
5'-GACGTAT AAAACGTT-3'
3'-CTGCAT ATTTTGCAA-5'
Thus, the plasmid has the optimal site of PcsI:
5'-A(5mC)GNNNNNNN(5mC)GT-3'
3'-TG(5mC)NNNNNNNG(5mC)A-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.
|
PcsI activity assay on DNA pMHpySE49/DriI
Lanes:
1 and 7 1 Kb SE DNA-markers
2 Control pMHpySE49/DriI
3 0.5 μl Pcs I (diluted 1/10)
4 1 μl Pcs I (diluted 1/10)
5 2 μl Pcs I (diluted 1/10)
6 1 μl Pcs I concentrated
Products were separated in 1,0% agarose gel in Buffer TAE
|
 |
| 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 | 50 - 75 | 0 - 10 | 10 - 25 | 50 - 75 | 50 |
|
| Reaction buffer | SE-buffer PcsI |
| Optimal temperature | 37oC |
| Storage conditions | 15 mM Tris-HCl (pH 7.5); 250 mM NaCl; 0,1 mM EDTA;
0,05% Triton X-100, 7 mM 2-mercaptoethanol;
100 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 10 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 PcsI |
| Methylation sensitivity | The enzyme cleaves only 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., 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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