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. 1978 Apr;75(4):1773–1777. doi: 10.1073/pnas.75.4.1773

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

N Patrick Higgins *, Craig L Peebles †, Akio Sugino *,‡, Nicholas R Cozzarelli *,†
PMCID: PMC392422  PMID: 347446

Abstract

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 ϕX174 DNA directed by nalAr mutants. Subunit B contains a 95,000-dalton polypeptide and is controlled by the gene specifying sensitivity to novobiocin (cou) because cour mutants produce a novobiocin-resistant subunit B and novobiocin-resitant 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, Km for 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.

Keywords: DNA supercoiling, DNA relaxation, novobiocin, nalidixic acid, DNA topoisomerase II

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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