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.
Topoisomerase I Hi-Loading Inhibition Kit
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.
Human-Topoisomerase-I-Hi-Load-Kit
References
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
https://dl.dropboxusercontent.com/u/6593368/ja412350f_s.pdf
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
https://www.ncbi.nlm.nih.gov/pubmed/?term=Proc.+Natl++Acad.+Sci.%2C+USA+99%3A+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
https://www.ncbi.nlm.nih.gov/pubmed/?term=Cancer+Research+60%3A+4538-4543