Description
Recognition site and hydrolysis position:
| G(5mC)G(5mC)NG(5mC)G(5mC) | G(5mC)G(5mC)↑NG(5mC)G(5mC) |
| (5mC)G(5mC)GN(5mC)G(5mC)G | (5mC)G(5mC)GN↓(5mC)G(5mC)G |
Source: An E.coli strain that carries the cloned Mte I gene from Microbacterium testaceum 17B
Substrate specificity:
Unit definition: One unit is defined as the amount of enzyme required to hydrolyze completely 1 μg of linearized plasmid pHspAI10/DriI+M.Fsp4HI in 1 hour at 55°C in a total reaction volume of 50 μl.
Assayed on: pHspAI10/DriI/metM.Fsp4HI is a plasmid pHspAI10, which is linearized with DriI, and, additionally, modified with Fsp4HI DNA methyltransferase. pHspAI10 carries a gene of HspAI DNA methyltransferase, that modifies the sequence 5`-GCGC-3`, producing 5`-G(5mC)GC-3`.
M.Fsp4HI modifies the sequence 5`-GCNGC-3`, producing 5`-G(5mC)NGC-3`.
A substrate pHspAI10/DriI additionally methylated with M.Fsp4HI includes one site 5`-G(5mC)G(5mC)NG(5mC)G(5mC)-3`/3`-(5mC)G(5mC)GN(5mC)G(5mC)G-5`, which is MteI canonical site [1]. The enzyme activity depends on a number and positions of methylated nucleotides in the recognition sequence. For example, MteI cuts the recognition site with six 5-methylcytosines, but the enzyme activity is reduced for more that one order [1].
Optimal SE-buffer: SE-buffer W
Enzyme activity (%):
| B | G | O | W | Y | ROSE |
| 25 | 75 | 75 | 100 | 50 | 100 |
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.
Ligation: –
Non-specific hydrolisis: No detectable degradation of 1μg of Lambda DNA was observed after incubation with 40 units of enzyme for 16 hours at 55°C in a total reaction volume of 50 μl.
Reagents Supplied with Enzyme
10 X SE-buffer W, DNA pHspAI10/DriI additionally methylated with M.Fsp4HI.
Methylation sensitivity: The enzyme cleaves C5-methylated DNA and doesn’t cut unmodified DNA.
Notes: References:
1.Degtyarev S.Kh., Rechkunova N.I., Zernov Yu.P., Dedkov V.S., Chizikov V.E., Van Calligan M., Williams R., Murray E. Bsp24I, a new unusual restriction endonuclease. // Gene, No.131, 93-95 (1993).
1.V.A. Chernukhin, E.V. Kileva, V.A. Sokolova., D.A. Gonchar, L.N. Golikova, V.S. Dedkov, N.A. Mikhnenkova, S.Kh. Degtyarev A new methyl-directed site-specific DNA endonuclease MteI cleaves nine nucleotides sequencer
5’-G(5mC)G(5mC)^NG(5mC)GC-3’/3’-CG(5mC)GN^(5mC)G(5mC)G-5’r
// Translated from “Ovchinnikov bulletin of biotechnology and physical and chemical biology” V.8, No 1, pp 16-26, 2012
