Topoisomerase assays

Abstract

Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are for assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA, and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay to examine topoisomerase covalent complexes in vivo, and an assay for measuring DNA cleavage in vitro.

Figure 3.3.1

Reactions of DNA topoisomerases. DNA topoisomerases catalyze the interconversion of different topological forms of DNA, such as the knotting and unknotting of DNA and catenation and decatenation of DNA rings. Type I topoisomerases are able to unknot or decatenate single-stranded knots and catenanes, but are unable to carry out these reactions on intact double-stranded DNA. (A) Both type I and type II topoisomerases can relax supercoiled DNA. Type II topoisomerases are able to carry out (B) the knotting/unknotting of intact double stranded DNA and (C) the catenation/decatenation of intact double-stranded DNA.

Figure 3.3.2

Gels obtained from topoisomerase I activity assays. Yeast topoisomerase I was purified from yeast cells expressing wild type yeast Top1 from the Gal1 promoter. A. Lane λ, λ HindIII molecular weight markers; lane S, 0.1 μg pUC18 plasmid with no added protein, other lanes contain decreasing amounts of purified protein. Samples were electrophoresed on a 1.0% agarose gel. B. The same conditions as in panel A, lane 4 were used, but increasing concentrations of ethidium bromide were added (from 100 ng/ml to 2 μg/ml ethidium bromide). Ethidium bromide is not an inhibitor of Top1, but because it intercalates and unwinds DNA, carrying out the reaction in the presence of ethidium bromide introduces positive supercoils in the DNA, which can be relaxed by Top1. Therefore one might erroneously conclude that ethidium bromide inhibits Top1. Approaches to distinguish effects of intercalation are discussed in detail by Bailly (Bailly, 2001).

Figure 3.3.2

Gels obtained from topoisomerase I activity assays. Yeast topoisomerase I was purified from yeast cells expressing wild type yeast Top1 from the Gal1 promoter. A. Lane λ, λ HindIII molecular weight markers; lane S, 0.1 μg pUC18 plasmid with no added protein, other lanes contain decreasing amounts of purified protein. Samples were electrophoresed on a 1.0% agarose gel. B. The same conditions as in panel A, lane 4 were used, but increasing concentrations of ethidium bromide were added (from 100 ng/ml to 2 μg/ml ethidium bromide). Ethidium bromide is not an inhibitor of Top1, but because it intercalates and unwinds DNA, carrying out the reaction in the presence of ethidium bromide introduces positive supercoils in the DNA, which can be relaxed by Top1. Therefore one might erroneously conclude that ethidium bromide inhibits Top1. Approaches to distinguish effects of intercalation are discussed in detail by Bailly (Bailly, 2001).

Figure 3.3.3. Topoisomerase II decatenation assay

Different amounts of purified yeast topoisomerase II were added to reactions that contained 0.2 μg kinetoplast DNA as a substrate. Lane M, λ HindIII molecular weight markers; lane S, substrate DNA; lane 1, substrate and 5 ng purified yeast topoisomerase II; lane 2, substrate and 50 ng purified yeast topoisomerase II; lane 3, substrate and 100 ng purified yeast topoisomerase II; lane 4, substrate and 200 ng purified yeast topoisomerase II; lane 5, substrate and 500 ng purified yeast topoisomerase II; lane 6, substrate and 1 μg purified yeast topoisomerase II. The fluorescence just below the well in lane S is due to the catenated kinetoplast DNA that fails to enter the gel; the decatenated product is clearly seen in lanes 1 to 5. Samples were electrophoresed on a 0.8% agarose gel.

Figure 3.3.4. ICE assay of Top2 covalent complexes

HeLa cells were treated with various etoposide concentrations for 1 hr. The ICE assay was performed as described in Basic protocol 3, and the slot blots of DNA recovered from the pellet after CsCl centrifugation, probed with an antibody directed against Top2 α is shown in panel A. The signal arises from Top2 that covalently associates with DNA. Panel B shows a quantitation of the signal from the blot shown in panel A.

Figure 3.3.5. Schematic representation of in vitro cleavage of a double-stranded oligonucleotide by topoisomerase I

The annealed oligonucleotides are shown; “*L” indicates the cordycepin label. Note that only the top strand is labeled. Upon addition of topoisomerase I and camptothecin, a specific cleavage by topoisomerase I occurs. The oligonucleotide is designed so that cleavage occurs specifically at the site indicated by the arrow. The topoisomerase I protein is trapped on the DNA upon addition of SDS, and the strands are separated by the addition of formamide. If no topoisomerase I cleavage occurs, the only product observed on a sequencing gel is the 37-nucleotide labeled substrate (the top strand). Topoisomerase I cleavage results in a smaller oligonucleotide. Note that the oligonucleotide that has protein covalently bound is unlabeled, so it will not be detected.

Figure 3.3.6. Plasmid DNA cleavage with purified Top2

The plasmid cleavage assay was carried out with 2 μg of purified yeast Top2 (a very high concentration). λ HindIII markers, Sc, substrate with no added protein, L, pUC18 linearized with a restriction enzyme, and increasing concentrations of etoposide as indicated on the figure. Note that a band that has the same electrophoretic mobility as linear DNA is absent without etoposide, but clearly seen when 1 μg/ml of etoposide is added. The linear DNA is seen with 3 μg/ml etoposide, and decreases in intensity as more etoposide is added. Samples containing ≥ 3 μg/ml of etoposide show smearing of DNA that arises from cleaving the DNA at multiple sites. This smearing is readily interpretable when purified Top2 is used, however care must be taken with crude enzyme preparations that may contain contaminating nuclease activity.

Figure 3.3.7. DNA cleavage with purified Top2

DNA cleavage assays were carried out using ³²P end labeled pUC18 DNA. Cleavage was carried out in the absence of inhibitor (lane marked 0), in the presence of 0.2 μg/ml mAMSA or 1.0 μg/ml mAMSA. Enzyme concentrations added ranged from 2 units (50 ng) to 25 units (1.25 μg).