Topoisomerase assays

Two DNA topoisomerases VI have been purified to homogeneity from archaeal strains. The first purification reported was that of the enzyme from Sulfolobus shibatae, an extremely thermophilic crenoarchaeota with an optimal growth temperature of 85°. This article presents the purification of the DNA topoisomerase VI from Pyrococcus furiosus, an extremely thermophilic anaerobic euryarchaeota with an optimal growth temperature of 95°. These two enzymes share many properties and can be purified by a similar procedure. However, the purification of the DNA topoisomerase VI from P. furiosus has been simplified and some modifications, required by the extreme thermophily of this enzyme, have been introduced into DNA topoisomerase assays. Finally, physical and enzymatic properties of DNA topoisomerase VI are discussed. 

Panigrahi, G. Zhao, B. Krepinsky, J. J. Sadowski, P. D. Toward a mechanism-based fluorescent assay for site-specific recombinases and topoisomerases Assay design and syntheses of fluorescent substrates. 

Identification of proteins that bind to Z-DNA added one further step to the establishment of the presence of Z-DNA in vivo and its possible biological role. Herbert and Rich [22] demonstrated an in vitro assay system where one type of double-stranded RNA adenosine deaminase, called DRAD-binding Z-DNA. There are evidences that topoisomerase II from Drosophila, hiunan and calf thymus recognizes a number of DNA shapes, including Z-DNA [34,35]. Bloomfield and coworkers [36] have found that the condensation of plasmids is enhanced by Z-DNA conformation in d(CG)n repeats. The information related to B-Z transition [31], the effect of ligands on it [28,29] and X-ray crystal structure data [37,38] appear to suggest that the possible biological role of this polymorphic form of DNA will be soon established. 

Taxonomically close to the Annonaceae, the Lauraceae family abounds with apor-phinoid alkaloids. A remarkable advance in the search for topoisomerase inhibitors from Lauraceae has been provided by Woo et al. (6). Using DNA-unwinding assay and structural modeling, they showed that dicentrine can attain a relatively planar conformation and molecular bulk which allow it to occupy the active site of topoisomerase II which becomes inactive. The requirement of a suboptimal conformation to achieve DNA binding appears to make dicentrine less potent against topoisomerase II than the... 

Shroyer KR, Homer P, Heinz D, et al. Validation of a novel immunocytochemical assay for topoisomerase II- and minichromosome maintenance protein 2 expression in cervical cytology. Cancer 2006 108 324-330. 

The low torsion constant at a = —0.025 is very similar to that observed in a supercoiled pBR322 that was partially relaxed by saturation binding of Escherichia coli single-strand binding (ssb) protein, and which persisted for over a month.(56) It is also similar to that recently inferred from an in vivo assay based on variation in repression efficiency with size of a putative DNA loop.(234) Indeed, it appears that anomalously low torsion constants may be universally encountered in the course of either partial or complete relaxation of supercoiled DNAs, regardless of whether the superhelix density is reduced by action of topoisomerase I, binding of ssb protein, binding of intercalated... 

Ruling out intercalation as a critical recognition mode proved harder on a spectroscopic basis. Thus resort was made to a biochemical Topoisomerase I assay. This assay, which sapphyrin passed in a negative sense, can be used to detect the unwinding that is considered diagnostic of intercalation. ... 

At Virginia Tech bioassays were carried out using four different yeast strains, obtained from BMS Pharmaceutical Research Institute, and designed to detect potential anticancer agents that act as inhibitors of the enzymes topoisomerase I or topoisomerase II, or as cytotoxic agents by some other mechanism. Because of the use of yeasts as the assay organism,... 

Gorczyca, W, Melamed, M. R., and Darzyrikiewicz, Z. (1993) Apoptosis of S-phase HL-60 cells induced by topoisomerase inhibitors detection of DNA strand breaks by flow cytometry using the in situ nick translation assay Toxicol Lett 67, 249-258. 

Lemer, C. G. and Saiki, A. Y. (1996) Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculo-virus-infected insect cells. Anal. Biochem. 240, 185-196. 

Modifications of the standard battery may be necessary for some classes, e.g., antibiotics which are toxic to bacteria or e.g., for compounds like topoisomerase inhibitors which interfere with the mammalian cell replication system. A selection of additional assays is being proposed, further modifications may be acceptable via discussion in the ICH Maintenance Process. Alternative strategies may consider assays like the in vivo Comet assay (single cell gel electrophoresis measuring DNA strand breaks) or gene mutation tests with transgenic animals or in vivo DNA adduct studies. 

The ability of metal complexes to unwind DNA has been put forth as an important criterion for proving an intercalative binding mode and has been observed with other complexes of phen, dppz, and phi (21, 28,30,33). The enzyme topoisomerase can be used to determine if small molecules unwind DNA, according to published procedures (28). We find that by using this assay, Ru(tpy)(dppz)OH22+ unwinds DNA by 17°, which is consistent with intercalative binding. 

Onishi et al. (1993) developed an assay that monitors the relaxation of super-coiled plasmid DNA induced by DNA topoisomerase. Supercoiling of DNA is important in the formation of chromatin and in determining functions of DNA, including replication, transcription, recombination, and repair. 

The reaction mixture contained 50 mM Tris-HCl buffer (pH 7.5), 120 mM KC1,10 mM MgCfe, 0.5 mM EDTA, 0.5 mM dithiothreitol, 30 /xg/mL bovine serum albumin, and 0.2 fig of pBR329 DNA. After incubation at 37°C for 30 minutes, the reaction was terminated by the addition of Sarkosyl in a final concentration of 1%. The reaction mixture was further incubated at 37°C for 30 minutes after proteinase K had been added to give a concentration of 50 /xg/mL. Assays were linear with up to 0.4 fig of topoisomerase I, and with time until about 95% of the substrate had been converted into relaxed DNA. 

Several ellipticines and 7//-pyrido[4,3-c]carbazoles were examined for their effect on topoisomerase I and II from trypanosomes (Table VI) (163). The activity of 9-bromoellipticine on topoisomerase II is especially interesting since it is not a DNA intercalator. Several other bis-7//-pyrido[4,3-c]carbazoles were strongly active in this assay. As indicated in Table VII, a study of the cytotoxicity and uptake by TBL CL2 mouse sarcoma cells of several oxazolopyridocarbazoles... 

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