Isolation and characterization of BsuE methyltransferase, a CGCG specific DNA methyltransferase from Bacillus subtilis.
The DNA methyltransferase M-BsuE that recognizes the sequence 5'-CGCG-3' has been isolated from Bacillus subtilis strain ISE15. A 1600-fold purification of M-BsuE was achieved by column chromatography on phosphocellulose, heparin-Sepharose, and DEAE-Sepharose. DNA methyltransferase activity was monitored in the column eluants radiochemically by the transfer of tritiated methyl groups from radiolabeled S-adenosylmethionine to poly(dGdC)-poly(dGdC) DNA, a sensitive and specific substrate for M-BsuE activity. The DNA sequence specificity of this methyltransferase activity was confirmed enzymatically by demonstrating that M-BsuE-methylated DNA was selectively protected from cleavage by the restriction enzyme isoschizomers, ThaI and FnuDII. Purified M-BsuE has an apparent molecular size of 41,000-43,000 as determined by gel filtration and migrates as a 41-kDa protein in a sodium dodecyl sulfate-polyacrylamide gel. DNA methylation by M-BsuE is dependent upon the presence of S-adenosylmethionine and 2-mercaptoethanol. M-BsuE methyltransferase activity is optimal at 37 degrees C in the presence of 50 mM Tris-HCl, pH 7.8, 25 mM KCl, 6 microM S-adenosylmethionine, 5 mM 2-mercaptoethanol, and 10 mM EDTA. M-BsuE methylates the external cytidine in its recognition sequence in both linear and supercoiled DNA. A unique property of M-BsuE is its ability to methylate 5'-CGCG-3' in Z-DNA.
D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D015257:DNA-Cytosine Methylases; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D009994:Osmolar Concentration; D013379:Substrate Specificity
C055352:DNA modification methylase BsuE; D015257:DNA-Cytosine Methylases; D008780:Methyltransferases