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Isolation and characterization of BsuE methyltransferase, a CGCG specific DNA methyltransferase from Bacillus subtilis. by M L Gaido; C R Prostko; J S Strobl is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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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

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Author

M L Gaido; C R Prostko; J S Strobl

Publisher

Elsevier BV

Published

1988

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)