DNA Gyrase in vitro activity

Studying DNA Gyrase in vitro activity

Investigations of in vitro reaction rates by gel electrophoresis and fluorescence spectroscopy

Research Project at:

Department of Molecular Cell Physiology
Faculty of Biology
Vrije Universiteit
De Boelelaan 1087
1081 HV Amsterdam (The Netherlands)
Supervisor: Dr Jacky L. Snoep

Related Projects

Hierarchical Control Analysis

Gyrase supercoiling activity

DNA Gyrase is a type II topoisomerase that catalyses transient doublestrand breaks and uses free energy of ATP hydrolysis for introducing negative supercoils into DNA molecules by reducing the helix-linking number in steps of 2. The opposing activities of Gyrase and topoisomerase I play a central role in determining the in vivo supercoiling level in prokaryotic cells (Drlica, 1992; Luttinger, 1995). They are thought to be involved in the regulation of gene expression depending on the free energy metabolism (van Workum, 1996). It was shown that both Gyrase activity in vitro and DNA supercoiling mediated by Gyrase and topoisomerase I in vivo depend on ATP/ADP ratios (van Workum, 1996; Westerhoff, 1988) and that DNA supercoiling influences the expression of Gyrase and topoisomerase I in E.coli (Menzel and Gellert, 1983; Tse-Dinh, 1985).

Approaches to a more detailed study on the control of topoisomerases on DNA supercoiling in vivo require data about kinetic properties of these enzymes, which are difficult to define in terms of traditional enzyme kinetics. This is because topoisomerases do not convert any substance A into a chemically distinct substance B. They introduce/ release tense into/ from DNA molecules by trapping conformational changes through resealing of transient breaks, thus convert several A_X into several A_X±Y. Gyrase also shows a so called slippage effect, that means, ATP hydrolysis is coupled to supercoiling activity to a rather variable extent (Westerhoff, 1988).

a 3D-Model of DNA Gyrase

DNA plasmids incubated with Gyrase for increasing periodes of time (f.l.t.r.) as visualized after electrophoresis on an agarose-gel (strong bands on top are open- circular plasmid molecules)

References

Bates AD, Maxwell A (1993)(In Focus) DNA Topology. Oxford: Oxford University Press.

Drlica K (1992) Control of bacterial supercoiling. Molecular Microbiology6: 425-433

Higgins CF, Dorman CJ, Stirling DA, Waddell L, Booth IR, May G, Bremer E (1988) A Physiological Role for DNA Supercoiling in the Osmotic Regulation of Gene Expression in S. typhimirum and E. coli. Cell52: 569-584.

Luttinger A (1995) The twisted 'life' of DNA in the cell: bacterial topoisomerases. Molecular Microbiology15: 601-606.

Menzel R and Gellert M (1983) Regulation of the genes for E. coli DNA Gyrase: homeostatic control of DNA supercoiling. Cell34: 105-113.

Tse-Dinh Y (1985) Regulation of the DNA topoisomerase 1 gene by DNA supercoiling. Nucleic Acids Research13: 4751-4763.

van Workum M, van Dooren SJM, Oldenburg N, Molenaar D, Jensen PR, Snoep JL, Westerhoff HV (1996) DNA supercoiling depends on the phosphorylation potential in E. coli. Molecular Microbiology20: 351-360.

Westerhoff HV, O'Dea M, Maxwell A, Gellert M (1988) DNA Supercoiling by DNA Gyrase - A Static Head Analysis. Cell Biophysics12: 157-181.