SDS-K In Vivo

Kit Description:
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This kit contains reagents to trap and detect covalent topoisomerase/DNA intermediates that form in vivo (for example inside cells or nuclei with endogenous topoisomerase).

Kit Contents: (volumes given for 100 assay kit size).

Camptothecin (10 mM)
Etoposide (10 mM)
10X TKE wash buffer.
2.5M KCl.
Cellular Lysis Solution.r

A Detailed Manual for the SDS-K+ Precipitation along with controls.

This TopoGEN kit is designed to quantify formation of the endogenous covalent protein/DNA intermediates that form with genomic DNA. Detecting the complex is greatly enhanced by topo poisons that stabilize the reaction (covalent) intermediate. Since the genomic DNA is radioactively tagged ([³H]-TdR), the procedure is quantitative and can be used to compare or identify different inhibitors in situ. Radioactivity is not supplied; however, detailed instructions are included which describe the procedure (for use with cell lines).

Shipping and Recommended Storage Conditions:

This kit is shipped on Dry Ice and should be stored at -20^o C.

The following materials and reagents are not supplied with this kit but will be required:

95% and 70% Ethanol

Glass Fiber Filters (used for TCA precipitations, such as Whatman GF/C)

Filtration manifold or single Millipore Filter stack.

alpha-[³H]-Thymidine

Scintillation vials, Scintillation fluid and counter

Background:

Both type I and II topoisomerases can be trapped in a covalent complex with DNA by adding protein denaturants (alkali, acid or detergents) while the enzyme is actively engaged in breaking and resealing steps on DNA. Although the detailed mechanism of trapping by denaturation is not known, it is generally accepted that capturing the covalent intermediate or "cleavable" complex is difficult because the intermediate has a relatively short life time. Thus, with normal amounts of topo I or II (i.e., sufficient enzyme to observe catalysis), detecting the covalent complex by denaturation requires the presence of topo inhibitors which tend to extend the life time of the cleavage complex. (Strictly speaking, these types of "inhibitors" are in fact better described as "poisons".) Formation of a covalent intermediate can be exploited as a means of identifying novel topo poison since the complex is a functional intermediate of the catalytic cycle of topo. Covalent topo/DNA complexes are generated by adding SDS to a reacon in which the enzyme is nicking and resealing DNA; a fraction (typically <5%) of the topo is trapped in a covalent complex with DNA. SDS binds only protein (not DNA) and non-covalently bound topo is irreversibly inactivated. Upon addition of KCl, insoluble crystals of potassium dodecyl sulfate form (we refer to this as SDS-K+) which co-precipitate free protein and only those DNAs covalently bound to topo I. If the DNA is labeled, the amount of label in the precipitate is a measure of DNA molecules with covalently bound (denatured) topo. DNA in the SDS-K+ precipitate can be recovered and shown to be all nicked (topo I reactions) or a mixture of nicks and double strand breaks (topo II), as expected from what we know of the reaction mechanism. The SDS-K+ method is particularly useful for quantifying topo DNA covalent complexes to: 1) identify topo I or II inhibitors or "poisons" and 2) to facilitate comparison of different inhibitors in their ability to increase the life time of the cleavage complex.

The diagram below shows the topo I reaction mechanism and the effect of termination of the reaction with SDS, followed by selective precipitation of covalent complexes

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