Topo I Drug Screening Kit 1018

Kit Description:
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Topoisomerase I introduces transient nicks in DNA at specific sites. Detection of these transient DNA nicks requires trapping the enzyme on DNA in a nicked intermediate complex using protein denaturants. The resulting covalent DNA/topo I complexes contain nicked open circular DNA which can be detected by agarose gel electrophoresis (with ethidium bromide). Trapping nicked intermediates is relatively inefficient; however, inhibitors, such as the natural product camptothecin, stabilize the intermediate and lead to an increase in the nicked DNA product. This forms the basis for a mechanistic drug screen designed to allow detection of agents that affect topoisomerase I by stabilizing the cleaved intermediate complex. The TopoGEN Topo I Drug Screening Kit is designed to allow the investigator to quickly identify novel inhibitors of topoisomerase I. The kit will allow detection of novel compounds that either stabilize the nicked intermediate or otherwise inhibit catalytic activity of topoisomerase I.

Kit Contents: (representative for 100 assay kit size).

-Supercoiled plasmid substrate DNA, 25 µg.

-Relaxed and nicked plasmid DNA markers.

-10X Topoisomerase I assay/cleavage buffer, 300 ul.

-Sodium dodecyl sulfate (SDS) termination buffer (10%).

-10X gel loading dye.

-Control inhibitor (Camptothecin; lyophilized).

-Detailed instruction manual.

Shipping and Recommended Storage Conditions:

This kit is shipped at ambient temperature; store at -20oC.

*Note: Enzyme sold separately, Go to Topo I

*The kits are designed to work with TopoGEN Human topoisomerase I (purchased separately from the kit; see product summary and price list enclosed). Since cleavage analyses require more enzyme than catalytic assays, TopoGEN sells the enzyme in larger lots which takes this difference into account.

References:

Trask et al., EMBO J. 3: 671-676 (1984)

Hsiang et al. J. Biol. Chem 260:14873-14878 (1985)

Use of the Topoisomerase I Drug Kit

Reactions indicated above each lane were carried out using 5 units of TopoGEN type I topoisomerase. The reactions were incubated for 30 min at 37 C and stopped with SDS. After digestion with proteinase K (50 ug/ml, 60 min at 37 C), samples were loaded onto a 1% agarose gel. Under these conditions, with excess topo I and camptothecin, significant conversion of supercoiled (form I) DNA to open circular (OC DNA) was observed. It is important to recognize that cleavage assays will consume more enzyme than catalytic (relaxation type) assays. This is because each cleavage event consumes one enzyme molecule; i.e., the reaction requires stoichiometic amounts of topo I.