Characterization of a restriction enzyme from Escherichia coli K carrying a mutation in the modification subunit.
The restriction enzyme from a restriction and modification-deficient strain of Escherichia coli K mutated in the modification gene (hsdM) has been purified using an in vitro complementation assay with a mutant restriction enzyme from a strain lacking only restriction. The restriction enzyme from the hsdM mutant lacks all of the activities that are associated with the wild type enzyme: binding of unmodified DNA to filters, cleavage, or methylation of unmodified DNA and ATP hydrolysis. It is shown that the enzyme from this hsdM mutant cannot bind S-adenosylmethionine, an allosteric effector in the restriction reaction. In the absence of enzyme activation by S-adenosylmethionine, no binding to unmodified DNA takes place. A comparison with other mutant restriction enzymes allows us to outline the biochemical role of the subunits of the E. coli K restriction endonuclease.
D004262:DNA Restriction Enzymes / Q000235:genetics* / Q000302:isolation & purification; D003851:Deoxyribonucleases / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology*; D005796:Genes; D007700:Kinetics; D046911:Macromolecular Substances; D009154:Mutation; D011485:Protein Binding
D046911:Macromolecular Substances; D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes