This kit is designed to allow the customer to screen for agents (drugs, natural products, small molecules, synthetics) that block the catalytic activity of human topoisomerase I (topo I). The use of high stoichiometric ratios of topo I:DNA induces cleavage complexes without the need to use topo I poisons (camptothecin) which facilitates detection of catalytic inhibitors of topoI. The assay (see ref 1) is based on the finding that topo I acts in a cooperative manner on DNA template, whereby it tends to display clustered activity (at neighboring sites, see ref 3); thus, at high ratios of enzyme:DNA, it is relatively easy to detect nicking by topo I in the absence of topo I poisons like camptothecin and its various congeners.
1. pHOT1 DNA, supercoiled substrate. Concentration of 0.25ug/ml (25 ug pHOT1 DNA in 100 ul TE buffer, 10mM Tris-HCl, pH7.5, 1 mM EDTA)
2. Marker DNA, Relaxed pHOT1; 0.05 ug/ul in 1x gel loading buffer (50 ul, load 2 ul as marker).
3. Topo I reaction buffer (10x TE; 300 ul) TE Buffer (1x) is 10 mM Tris-HCl pH 7.9, 1 mM EDTA
4. 10% Sodium Dodecyl Sulfate (300 ul): To terminate reactions, use 0.1 volume (final 1%).
5. 10x gel loading buffer (300 ul): 0.25% bromophenol blue, 50% glycerol, use 0.1 volume in reactions.
6. Proteinase K (500 ul) at 0.5 mg/ml. This is a 10x stock of proteinase K.
7. Purified human topoisomerase I provided at 25 units/ul (20ul). A total 500 units is included for Cat TG1017-1 and 1000 units for Cat TG1017-2.
The kit will be shipped on dry ice. Upon receipt, the DNA should be stored at 4° C and the buffers and enzyme stored at -20° C. Avoid frequent freeze/thaw cycles with the plasmid as this may contribute to DNA breakage and enzyme inactivation.
1. Akerman, K., Fagenson, A., Cyril, V., Taylor, M., Muller, M, Akerman, M., and Munro O. (2014) Gold(III) macrocycles: nucleotide-specific unconventional catalytic inhibitors of human topoisomerase I. (2014) JACS: 136:5670-5682
2. Mao, Y., Mehl, I., and Muller, M.T. (2002) Nuclear and Nucleolar Localization by the N-terminal Domain of DNA Topoisomerase I. Proc. Natl Acad. Sci., USA 99: 1235-1240
3. Mao, Y. , Okada, S., Chang, L. and Muller, M.T.p53 Dependence of Topoisomerase I Recruitment in vivo (2000) Cancer Research 60: 4538-4543