Sequence-specific BamHI methylase. Purification and characterization.
BamHI methylase has been purified to apparent homogeneity. The isolated form of the enzyme is a single polypeptide with a molecular weight of 56,000 as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Unlike BamHI endonuclease, which is isolated as a dimer and higher aggregates, the methylase has no apparent higher form. The methylase requires S-adenosyl-L-methionine as the methyl-group donor and is inhibited by Mg2+. The enzyme is also inhibited by 2,3-butanedione and reagents specific for sulfhydryl groups, such as N-ethylmaleimide, which suggests a role for arginine and cysteine residues, respectively. DNA efficiently protects the enzyme against the butanedione modification while S-adenosylmethionine has no effect. In contrast, S-adenosylmethionine protects against cysteine modification while DNA produces only small amounts of protection. Studies on the mechanism of methylation indicate that both strands of the recognition sequence are modified in a single binding event. The sequence specificity of the methylase is relaxed upon the addition of glycerol in the reaction mixture. In the presence of 30% glycerol the enzyme methylates sequences that are also recognized by BamHI endonuclease when acting under conditions of relaxed specificity.
D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D015257:DNA-Cytosine Methylases; D003931:Diacetyl / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism*; D008970:Molecular Weight; D013379:Substrate Specificity
C042525:DNA modification methylase BamHI; D015257:DNA-Cytosine Methylases; D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases; D003931:Diacetyl; D005990:Glycerol