Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Dec 15;24(24):4868–4873. doi: 10.1093/nar/24.24.4868

Probing the role of the ATP-operated clamp in the strand-passage reaction of DNA gyrase.

A P Tingey 1, A Maxwell 1
PMCID: PMC146357  PMID: 9016655

Abstract

The high-resolution structure of the 43 kDa N-terminal fragment of the DNA gyrase B protein shows a large cavity within the protein dimer. The approximate size of this cavity is 20 A, suggesting it could accommodate a DNA helix. Computer-modelling studies of this cavity suggest that it contains a constriction, reducing the width to approximately 13 A, principally caused by the side chain of Arg286. We have used site-directed mutagenesis to alter this residue to Gln. Gyrase bearing this mutation shows virtually no supercoiling activity and near-normal relaxation and DNA cleavage activities. The mutated protein has ATPase activity which cannot be stimulated by DNA. These data support the proposed role of the 43 kDa domain as an ATP-operated clamp which binds DNA during the supercoiling cycle. The lack of DNA-dependent ATPase of the mutant may indicate that binding of DNA within the clamp is a prerequisite for stimulation of the ATPase activity.

Full Text

The Full Text of this article is available as a PDF (193.7 KB).

Selected References

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

  1. Ali J. A., Jackson A. P., Howells A. J., Maxwell A. The 43-kilodalton N-terminal fragment of the DNA gyrase B protein hydrolyzes ATP and binds coumarin drugs. Biochemistry. 1993 Mar 16;32(10):2717–2724. doi: 10.1021/bi00061a033. [DOI] [PubMed] [Google Scholar]
  2. Bates A. D., Maxwell A. DNA gyrase can supercoil DNA circles as small as 174 base pairs. EMBO J. 1989 Jun;8(6):1861–1866. doi: 10.1002/j.1460-2075.1989.tb03582.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bates A. D., O'Dea M. H., Gellert M. Energy coupling in Escherichia coli DNA gyrase: the relationship between nucleotide binding, strand passage, and DNA supercoiling. Biochemistry. 1996 Feb 6;35(5):1408–1416. doi: 10.1021/bi952433y. [DOI] [PubMed] [Google Scholar]
  4. Berger J. M., Gamblin S. J., Harrison S. C., Wang J. C. Structure and mechanism of DNA topoisomerase II. Nature. 1996 Jan 18;379(6562):225–232. doi: 10.1038/379225a0. [DOI] [PubMed] [Google Scholar]
  5. Brookes S. T., Craig K. S., Cunnane S. C. Combined continuous flow isotope ratio mass spectrometry techniques for tracing the metabolism of 13C-labelled fatty acids. Biochem Soc Trans. 1994 May;22(2):164S–164S. doi: 10.1042/bst022164s. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Caron P. R., Wang J. C. Appendix. II: Alignment of primary sequences of DNA topoisomerases. Adv Pharmacol. 1994;29B:271–297. doi: 10.1016/s1054-3589(08)61143-6. [DOI] [PubMed] [Google Scholar]
  8. Corbett A. H., Zechiedrich E. L., Osheroff N. A role for the passage helix in the DNA cleavage reaction of eukaryotic topoisomerase II. A two-site model for enzyme-mediated DNA cleavage. J Biol Chem. 1992 Jan 15;267(2):683–686. [PubMed] [Google Scholar]
  9. Drlica K., Coughlin S. Inhibitors of DNA gyrase. Pharmacol Ther. 1989;44(1):107–121. doi: 10.1016/0163-7258(89)90093-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Gellert M., Mizuuchi K., O'Dea M. H., Itoh T., Tomizawa J. I. Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4772–4776. doi: 10.1073/pnas.74.11.4772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gilbert E. J., Maxwell A. The 24 kDa N-terminal sub-domain of the DNA gyrase B protein binds coumarin drugs. Mol Microbiol. 1994 May;12(3):365–373. doi: 10.1111/j.1365-2958.1994.tb01026.x. [DOI] [PubMed] [Google Scholar]
  13. Gormley N. A., Orphanides G., Meyer A., Cullis P. M., Maxwell A. The interaction of coumarin antibiotics with fragments of DNA gyrase B protein. Biochemistry. 1996 Apr 16;35(15):5083–5092. doi: 10.1021/bi952888n. [DOI] [PubMed] [Google Scholar]
  14. Hallett P., Grimshaw A. J., Wigley D. B., Maxwell A. Cloning of the DNA gyrase genes under tac promoter control: overproduction of the gyrase A and B proteins. Gene. 1990 Sep 1;93(1):139–142. doi: 10.1016/0378-1119(90)90148-k. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  17. Jackson A. P., Maxwell A. Identifying the catalytic residue of the ATPase reaction of DNA gyrase. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11232–11236. doi: 10.1073/pnas.90.23.11232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krasnow M. A., Cozzarelli N. R. Catenation of DNA rings by topoisomerases. Mechanism of control by spermidine. J Biol Chem. 1982 Mar 10;257(5):2687–2693. [PubMed] [Google Scholar]
  19. Kreuzer K. N., Cozzarelli N. R. Formation and resolution of DNA catenanes by DNA gyrase. Cell. 1980 May;20(1):245–254. doi: 10.1016/0092-8674(80)90252-4. [DOI] [PubMed] [Google Scholar]
  20. Lewis R. J., Singh O. M., Smith C. V., Skarzynski T., Maxwell A., Wonacott A. J., Wigley D. B. The nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray crystallography. EMBO J. 1996 Mar 15;15(6):1412–1420. [PMC free article] [PubMed] [Google Scholar]
  21. Maxwell A., Gellert M. The DNA dependence of the ATPase activity of DNA gyrase. J Biol Chem. 1984 Dec 10;259(23):14472–14480. [PubMed] [Google Scholar]
  22. Maxwell A. The interaction between coumarin drugs and DNA gyrase. Mol Microbiol. 1993 Aug;9(4):681–686. doi: 10.1111/j.1365-2958.1993.tb01728.x. [DOI] [PubMed] [Google Scholar]
  23. Maxwell A. The molecular basis of quinolone action. J Antimicrob Chemother. 1992 Oct;30(4):409–414. doi: 10.1093/jac/30.4.409. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Parker C. N., Halford S. E. Dynamics of long-range interactions on DNA: the speed of synapsis during site-specific recombination by resolvase. Cell. 1991 Aug 23;66(4):781–791. doi: 10.1016/0092-8674(91)90121-e. [DOI] [PubMed] [Google Scholar]
  26. Reece R. J., Maxwell A. DNA gyrase: structure and function. Crit Rev Biochem Mol Biol. 1991;26(3-4):335–375. doi: 10.3109/10409239109114072. [DOI] [PubMed] [Google Scholar]
  27. Reece R. J., Maxwell A. The C-terminal domain of the Escherichia coli DNA gyrase A subunit is a DNA-binding protein. Nucleic Acids Res. 1991 Apr 11;19(7):1399–1405. doi: 10.1093/nar/19.7.1399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Reece R. J., Maxwell A. Tryptic fragments of the Escherichia coli DNA gyrase A protein. J Biol Chem. 1989 Nov 25;264(33):19648–19653. [PubMed] [Google Scholar]
  29. Roca J., Wang J. C. DNA transport by a type II DNA topoisomerase: evidence in favor of a two-gate mechanism. Cell. 1994 May 20;77(4):609–616. doi: 10.1016/0092-8674(94)90222-4. [DOI] [PubMed] [Google Scholar]
  30. Roca J., Wang J. C. The capture of a DNA double helix by an ATP-dependent protein clamp: a key step in DNA transport by type II DNA topoisomerases. Cell. 1992 Nov 27;71(5):833–840. doi: 10.1016/0092-8674(92)90558-t. [DOI] [PubMed] [Google Scholar]
  31. Rádl S. Structure-activity relationships in DNA gyrase inhibitors. Pharmacol Ther. 1990;48(1):1–17. doi: 10.1016/0163-7258(90)90014-s. [DOI] [PubMed] [Google Scholar]
  32. Smart O. S., Goodfellow J. M., Wallace B. A. The pore dimensions of gramicidin A. Biophys J. 1993 Dec;65(6):2455–2460. doi: 10.1016/S0006-3495(93)81293-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Staudenbauer W. L., Orr E. DNA gyrase: affinity chromatography on novobiocin-Sepharose and catalytic properties. Nucleic Acids Res. 1981 Aug 11;9(15):3589–3603. doi: 10.1093/nar/9.15.3589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Sugino A., Peebles C. L., Kreuzer K. N., Cozzarelli N. R. 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. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4767–4771. doi: 10.1073/pnas.74.11.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tamura J. K., Bates A. D., Gellert M. Slow interaction of 5'-adenylyl-beta,gamma-imidodiphosphate with Escherichia coli DNA gyrase. Evidence for cooperativity in nucleotide binding. J Biol Chem. 1992 May 5;267(13):9214–9222. [PubMed] [Google Scholar]
  37. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1996;65:635–692. doi: 10.1146/annurev.bi.65.070196.003223. [DOI] [PubMed] [Google Scholar]
  38. Westerhoff H. V., O'Dea M. H., Maxwell A., Gellert M. DNA supercoiling by DNA gyrase. A static head analysis. Cell Biophys. 1988 Jan-Jun;12:157–181. doi: 10.1007/BF02918357. [DOI] [PubMed] [Google Scholar]
  39. Wigley D. B., Davies G. J., Dodson E. J., Maxwell A., Dodson G. Crystal structure of an N-terminal fragment of the DNA gyrase B protein. Nature. 1991 Jun 20;351(6328):624–629. doi: 10.1038/351624a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES