Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1981 Aug 11;9(15):3589–3603. doi: 10.1093/nar/9.15.3589

DNA gyrase: affinity chromatography on novobiocin-Sepharose and catalytic properties.

W L Staudenbauer, E Orr
PMCID: PMC327377  PMID: 6269086

Abstract

Novobiocin-Sepharose was prepared by coupling of novobiocin to Epoxy-activated Sepharose 6B and used as an affinity adsorbent. Four novobiocin-binding proteins were isolated from crude extracts of Escherichia coli with molecular weights of 105, 92, 85 and 40 kdal. The two larger proteins were identified as the A subunit (gyrA protein) and the B subunit (gyrB protein) of DNA gyrase topoisomerase II). By this method the two gyrase components can be easily separated and purified in high yield. Although both proteins are involved in the ATP-dependent supercoiling of relaxed plasmid DNA, only the gyrB protein is required for catalyzing the cleavage of ATP. The gyrB protein ATPase activity is competitively inhibited by novobiocin and related coumarin antibiotics. ATP hydrolysis is unaffected by the addition of either gyrA protein or DNA but stimulated in the presence of both.

Full text

PDF
3591

Images in this article

3594Image
on p.3594

Selected References

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

  1. Bouché J. P., Zechel K., Kornberg A. dnaG gene product, a rifampicin-resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form. J Biol Chem. 1975 Aug 10;250(15):5995–6001. [PubMed] [Google Scholar]
  2. Brown P. O., Peebles C. L., Cozzarelli N. R. A topoisomerase from Escherichia coli related to DNA gyrase. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6110–6114. doi: 10.1073/pnas.76.12.6110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cozzarelli N. R. DNA gyrase and the supercoiling of DNA. Science. 1980 Feb 29;207(4434):953–960. doi: 10.1126/science.6243420. [DOI] [PubMed] [Google Scholar]
  4. DIXON M. The determination of enzyme inhibitor constants. Biochem J. 1953 Aug;55(1):170–171. doi: 10.1042/bj0550170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Depew R. E., Liu L. F., Wang J. C. Interaction between DNA and Escherichia coli protein omega. Formation of a complex between single-stranded DNA and omega protein. J Biol Chem. 1978 Jan 25;253(2):511–518. [PubMed] [Google Scholar]
  6. Fairweather N. F., Orr E., Holland I. B. Inhibition of deoxyribonucleic acid gyrase: effects on nucleic acid synthesis and cell division in Escherichia coli K-12. J Bacteriol. 1980 Apr;142(1):153–161. doi: 10.1128/jb.142.1.153-161.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gellert M., Fisher L. M., O'Dea M. H. DNA gyrase: purification and catalytic properties of a fragment of gyrase B protein. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6289–6293. doi: 10.1073/pnas.76.12.6289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gellert M., Mizuuchi K., O'Dea M. H., Nash H. A. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3872–3876. doi: 10.1073/pnas.73.11.3872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gellert M., O'Dea M. H., Itoh T., Tomizawa J. Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4474–4478. doi: 10.1073/pnas.73.12.4474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Godfrey J. C., Price K. E. Structure-activity relationships in coumermycins. Adv Appl Microbiol. 1972;15:231–296. doi: 10.1016/s0065-2164(08)70094-7. [DOI] [PubMed] [Google Scholar]
  11. Higgins N. P., Peebles C. L., Sugino A., Cozzarelli N. R. Purification of subunits of Escherichia coli DNA gyrase and reconstitution of enzymatic activity. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1773–1777. doi: 10.1073/pnas.75.4.1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Klevan L., Wang J. C. Deoxyribonucleic acid gyrase-deoxyribonucleic acid complex containing 140 base pairs of deoxyribonucleic acid and an alpha 2 beta 2 protein core. Biochemistry. 1980 Nov 11;19(23):5229–5234. doi: 10.1021/bi00564a012. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lanzetta P. A., Alvarez L. J., Reinach P. S., Candia O. A. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979 Nov 15;100(1):95–97. doi: 10.1016/0003-2697(79)90115-5. [DOI] [PubMed] [Google Scholar]
  15. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  16. Mizuuchi K., O'Dea M. H., Gellert M. DNA gyrase: subunit structure and ATPase activity of the purified enzyme. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5960–5963. doi: 10.1073/pnas.75.12.5960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Orr E., Fairweather N. F., Holland I. B., Pritchard R. H. Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12. Mol Gen Genet. 1979;177(1):103–112. doi: 10.1007/BF00267259. [DOI] [PubMed] [Google Scholar]
  18. Orr E., Staudenbauer W. L. An Escherichia coli mutant thermosensitive in the B subunit of DNA gyrase: effect on the structure and replication of the colicin E1 plasmid in vitro. Mol Gen Genet. 1981;181(1):52–56. doi: 10.1007/BF00339004. [DOI] [PubMed] [Google Scholar]
  19. Ryan M. J. Coumermycin A1: A preferential inhibitor of replicative DNA synthesis in Escherichia coli. I. In vivo characterization. Biochemistry. 1976 Aug 24;15(17):3769–3777. doi: 10.1021/bi00662a020. [DOI] [PubMed] [Google Scholar]
  20. Spector T. Refinement of the coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0.5 to 50 microgram of protein. Anal Biochem. 1978 May;86(1):142–146. doi: 10.1016/0003-2697(78)90327-5. [DOI] [PubMed] [Google Scholar]
  21. Staudenbauer W. L. Letters to the editor: Novobiocin-a specific inhibitor of semiconservative DNA replication in permeabilized Escherichia coli cells. J Mol Biol. 1975 Jul 25;96(1):201–205. doi: 10.1016/0022-2836(75)90191-6. [DOI] [PubMed] [Google Scholar]
  22. Sugino A., Higgins N. P., Brown P. O., Peebles C. L., Cozzarelli N. R. Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4838–4842. doi: 10.1073/pnas.75.10.4838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sugino A., Higgins N. P., Cozzarelli N. R. DNA gyrase subunit stoichiometry and the covalent attachment of subunit A to DNA during DNA cleavage. Nucleic Acids Res. 1980 Sep 11;8(17):3865–3874. doi: 10.1093/nar/8.17.3865. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES