Rapid detection and isolation of covalent DNA/protein complexes: application to topoisomerase I and II.

Abstract

A rapid and simple method has been developed which allows detection and isolation of covalent DNA/protein adducts. The method is based upon the use of an ionic detergent, SDS, to neutralize cationic sites of weakly bound proteins thereby resulting in their dissociation off the helix. Proteins tightly or covalently bound to DNA that are not dissociable by SDS, result in the precipitation of the DNA fragment by the addition of KCl; however, free nucleic acid does not precipitate. The method is particularly useful as an analytical tool to titrate the binding of prototypic covalent binding proteins, topoisomerase I and II; thus, quantitation of topoisomerase activity is possible under defined conditions. As an analytical tool the method can be used as a general assay in the purification of as yet unidentified topoisomerases or other activities that bind DNA covalently. Moreover, the technology can be adapted for use in a preparative mode to separate covalent complexes from free DNA in a single step.

Stabilization of type I topoisomerase-DNA covalent complexes by actinomycin D.

“…The possibility that actinomycin D stabilized the covalent DNA-topoisomerase I intermediate was tested by using the KDodSO4 precipitation assay (15,16). KDodSO4 precipitates only those DNA molecules with covalently bound topoisomerase and thus measures the concentration of DNA molecules trapped with protein (16).…”

“…KDodSO4 precipitates only those DNA molecules with covalently bound topoisomerase and thus measures the concentration of DNA molecules trapped with protein (16). Reactions between topoisomerase I and simian virus 40 DNA were carried out in the presence or absence of the drug, NaDodSO4 was added, and the complexes were precipitated by addition of KCl to measure formation of covalent complexes.…”

“…The covalent intermediate is a functional reaction intermediate in the process of breaking and rejoining the DNA substrate (13)(14)(15), and we have shown that the amount of DNA trapped in a covalent complex with topoisomerase I is a quantitative measure of the steady-state levels of topoisomerase activity (16). Therefore, the relative activity of endogenous enzyme can be determined by measuring the ratio between covalently bound and free topoisomerase.…”

Reversal of MDR1-associated resistance to topotecan by PAK-200S, a new dihydropyridine analogue, in human cancer cell lines

“…Cells were harvested, washed and reincubated for 30 min at 37°C in [ 14 C]dThy-free medium containing 1.0-100 µM TPT with or without 2.0 µM PAK-200S. After drug exposure cells were washed in pre-warmed PBS (37°C) and the potassium chloride-SDS co-precipitation assay was immediately performed using 1 ml lysis buffer (1.25% SDS, 5 mM EDTA, 0.4 mg ml -1 sheared salmon sperm DNA, adjusted to pH 8.0) per 10 6 cells as described earlier (Trask et al, 1984;Vanhoefer et al, 1997). The results were calculated as percentage of precipitable [ 14 C]dThy-labelled DNA of drug-treated cells of total [ 14 C]dThy-DNA.…”

In Vitro Evolution of Preferred Topoisomerase II DNA Cleavage Sites

“…None of the contamination controls produced a band upon UV shadowing of the gel. K ϩ /SDS DNA Cleavage and Religation Assays-Levels of cleavage complex formation were monitored by the K ϩ /SDS precipitation assay (35,36). Oligonucleotide pools (from each round that was assayed) were digested with EcoRI and BamHI restriction endonucleases, and the ends were filled in using Klenow fragment and [␣- 32 P]dATP in the presence of nonradioactive nucleotides (37).…”