E. coli Topoisomerase IV Cleavage Assay Kits
These kits are designed specifically for cleavage reactions. They contain cleavage specific E. coli topo IV enzyme, supercoiled pBR322 DNA substrate, the Cleavage Assay and Dilution buffers as well as 10X GSTEB - our new stop and loading buffer.
Interruption of the DNA breakage-reunion step or stabilisation of the DNA-breakage step of the type II topoisomerase reaction can lead to cell death and is the mechanism by which the quinolones, such as nalidixic acid and ciprofloxacin, act. Cleavage assays are particularly useful in determining if a potential compound acts by this mechanism. Quinolones and similar compounds do not require ATP to stabilise the cleavage intermediate, thus the cleavage assay buffer does not contain ATP. However, if you are concerned that your compound/molecule of interest requires ATP to stabilise the cleaved intermediate, ATP can be added to the reaction.
Cleavage specific enzyme is available separately on request.
DNA Topoisomerase IV (Topo IV) is a bacterial type II topoisomerase which can decatenate and relax supercoiled DNA and it is a validated target for antibacterial compounds, such as the quinolones and the coumarins. Escherichia coli Topo IV is prepared by overexpressing the ParC and ParE subunits in E. coli and purifying them by methods adapted from Peng and Marians, 19991.
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- Peng, H. and Marians, K.J. (1999) Overexpression and purification of bacterial topoisomerase IV, in DNA Topoisomerase Protocols Vol. I (Bjornsti, M-A., and Osheroff, N. eds.), Humana Press, Totowa, N.Jersey pp.163-169