Mechanism of action of nalidixic acid: Purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme

Proceedings of the National Academy of Sciences of the United States of America - Tập 74 Số 11 - Trang 4767-4771 - 1977
Akio Sugino1, Craig L. Peebles2, Kenneth N. Kreuzer3, Nicholas R. Cozzarelli1,2,3
1Department of Biochemistry, The University of Chicago, Chicago, Illinois 60637
2Department of Biophysics and Theoretical Biology, The University of Chicago, Chicago, Illinois 60637
3Department of Committee on Genetics, The University of Chicago, Chicago, Illinois 60637

Tóm tắt

A target protein for nalidixic and oxolinic acids in Escherichia coli , the nalA gene product (Pnal), was purified to homogeneity as judged by gel electrophoresis, using an in vitro complementation assay. It is a dimer of identical 110,000-dalton subunits. A polypeptide of this molecular weight is uniquely induced by a λ nalA transducing phage, thereby showing that the purified Pnal is a product of the nalA gene. Nalidixic and oxolinic acids inhibit DNA gyrase activity and induce formation of a relaxation complex analogue. Treatment of the complex with sodium dodecyl sulfate causes a doublestrand break in the DNA substrate and the resulting linear molecule seems covalently bound to protein. Complex formation, unlike the introduction of supertwists, does not require ATP or relaxed circular DNA and is insensitive to novobiocin. DNA gyrase from a strain with a nalA mutation conferring drug resistance ( nalA r ) is 1/100 as sensitive to oxolinic and nalidixic acids with respect to inhibition of supertwisting and induction of the pre-linearization complex. Addition of Pnal restores drug sensitivity and stimulates DNA gyrase activity. DNA gyrase preparations and Pnal catalyze a third reaction sensitive to nalidixic and oxolinic acids, the ATP-independent relaxation of supertwister DNA. Relaxation by gyrase from nalA r cells is drug resistant. The nicking-closing activity is distinct from E. coli ω protein in several properties, including the ability to relax positively supertwisted DNA. We postulate that the nalA gene product occurs in two molecular forms, as Pnal and as a gyrase component. Both forms catalyze nicking-closing, and inhibition of this activity by nalidixic and oxolinic acids may account for the inhibition of DNA synthesis by these drugs.

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Proceedings of the National Academy of Sciences of the United States of America

Tập 74 Số 11

4767-4771

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