Partial purification of the Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase: in vitro methylation completely protects bacteriophage lambda deoxyribonucleic acid against cleavage by R-EcoRII.
A procedure is described for the partial purification of the deoxyribonucleic acid (DNA)-cytosine methylases controlled by the RII plasmid and by the Escherichia coli mec+ gene. The two enzymes exhibit similar but distinct chromatographic behavior on diethylaminoethyl-cellulose and phosphocellulose. Preliminary studies on the two methylases indicate that they are indistinguishable with respect to their Km for S-adenosylmethionine and their pH (in tris (hydroxymethyl)aminomethane buffer) and NaCl concentration optima. In vitro methylation of various phage lambda DNA substrates by the mec'r RII enzyme modifies the DNA to a form that is completely resistant to double-stranded cleavage by the RII restriction endonuclease (R-EcoRII). These results are consistent with our earlier proposal that the mec8ethylase recognizes RII host specificity sites.
Partial purification of the Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase: in vitro methylation completely protects bacteriophage lambda deoxyribonucleic acid against cleavage by R-EcoRII. A procedure is described for the partial purification of the deoxyribonucleic acid (DNA)-cytosine methylases controlled by the RII plasmid and by the Escherichia coli mec+ gene. The two enzymes exhibit similar but distinct chromatographic behavior on diethylaminoethyl-cellulose and phosphocellulose. Preliminary studies on the two methylases indicate that they are indistinguishable with respect to their Km for S-adenosylmethionine and their pH (in tris (hydroxymethyl)aminomethane buffer) and NaCl concentration optima. In vitro methylation of various phage lambda DNA substrates by the mec'r RII enzyme modifies the DNA to a form that is completely resistant to double-stranded cleavage by the RII restriction endonuclease (R-EcoRII). These results are consistent with our earlier proposal that the mec8ethylase recognizes RII host specificity sites.