Purification and properties of the Eco57I restriction endonuclease and methylase--prototypes of a new class (type IV).
The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.
Janulaitis
A
A
Institute of Biotechnology FERMENTAS, Vilnius, Lithuania.
Petrusyte
M
M
Maneliene
Z
Z
Klimasauskas
S
S
Butkus
V
V
D001483:Base Sequence; D004247:DNA; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D004247:DNA; C059642:DNA modification methylase Eco57I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes; C054219:endodeoxyribonuclease Eco57I