Purification of subunits of Escherichia coli DNA gyrase and reconstitution of enzymatic activity

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作者：

Higgins，N.，P.，Peebles，C.，L.，Sugino，A.，Cozzarelli，N.

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摘要：

Extensively purified DNA gyrase from Escherichia coli is inhibited by nalidixic acid and by novobiocin. The enzyme is composed of two subunits, A and B, which were purified as separate components. Subunit A is the product of the gene controlling sensitivity to nalidixic acid (nalA) because: (i) the electrophoretic mobility of subunit A in the presence of sodium dodecyl sulfate is identical to that of the 105,000-dalton nalA gene product; (ii) mutants that are resistant to nalidixic acid (nalAr) produce a drug-resistant subunit A; and (iii) wild-type subunit A confers drug sensitivity to in vitro synthesis of φ X174 DNA directed by nalArmutants. Subunit B contains a 95,000-dalton polypeptide and is controlled by the gene specifying sensitivity to novobiocin (cou) because courmutants produce a novobiocin-resitant subunit B and novobiocin-resistant gyrase is made drug sensitive by wild-type subunit B. Subunits A and B associate, so that gyrase was also purified as a complex containing 105,000- and 95,000-dalton polypeptides. This enzyme and gyrase reconstructed from subunits have the same drug sensitivity, Kmfor ATP, and catalytic properties. The same ratio of subunits gives efficient reconstitution of the reactions intrinsic to DNA gyrase, including catalysis of supercoiling of closed duplex DNA, relaxation of supercoiled DNA in the absence of ATP, and site-specific cleavage of DNA induced by sodium dodecyl sulfate.

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关键词：

Biochemistry DNA supercoiling DNA relaxation Novobiocin Nalidixic acid DNA topoisomerase II

DOI：

10.1073/pnas.75.4.1773

被引量：

658

年份：

1978