The binding of gyrase to DNA: analysis by retention by nitrocellulose filters (original) (raw)

Journal Article

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1

Department of Biochemistry, University of Chicago

Chicago, IL 60615

2

Department of Biochemistry, University of Wyoming

Laramie, WY 82071, USA

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1

Department of Biochemistry, University of Chicago

Chicago, IL 60615

3

Biophysics and Theoretical Biology, University of Chicago

Chicago, IL 60615

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Revision received:

27 September 1982

Accepted:

27 September 1982

Published:

11 November 1982

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N.P. Higgins, N.R. Cozzarelli, The binding of gyrase to DNA: analysis by retention by nitrocellulose filters, Nucleic Acids Research, Volume 10, Issue 21, 11 November 1982, Pages 6833–6847, https://doi.org/10.1093/nar/10.21.6833
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Abstract

Three distinet Escherichiacoli DNA gyrase complexes with DNA can be identified using a nitrocellulose filter–binding assay. One complex consists of an ensemble of two subunit A and two subunit Bprotomers bound non–covalently to specific sequences of DNA. High levels of each subunit alone are inactive but a single gyrase molecule binds DNA to a filter. At 23°, the complex has a dissociation constant of approximately 10 −10 M and a half-time of decay of about 60 h. It is sufficiently stable that it can be purified by gel filtration and retain full supercoiling activity. Gyrase binds preferentially to relaxed DNA over supercoiled DNA by a factor of about 10. On addition of oxolinic acid, a second complex is formed that is distinguished by its stability in high ionic strength solutions and by efficient conversion to a third form upon addition of protein denaturants. The first and second complexes require Mg ++ for optimal formation. The third form has been shown previously to contain denatured A protomers covalently linked to DNA that is broken at the site of attachment.

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