DNA cleavage assay for the identification of topoisomerase I inhibitors

Abstract

The inhibition of DNA topoisomerase I (Top1) has proven to be a successful approach in the design of anticancer agents. However, despite the clinical successes of the camptothecin derivatives, a significant need for less toxic and more chemically stable Top1 inhibitors still persists. Here, we describe one of the most frequently used protocols to identify novel Top1 inhibitors. These methods use uniquely 3′-radiolabeled DNA substrates and denaturing polyacrylamide gel electrophoresis to provide evidence for the Top1-mediated DNA cleaving activity of potential Top1 inhibitors. These assays allow comparison of the effectiveness of different drugs in stabilizing the Top1-DNA intermediate or cleavage (cleavable) complex. A variation on these assays is also presented, which provides a suitable system for determining whether the inhibitor blocks the forward cleavage or religation reactions by measuring the reversibility of the drug-induced Top1–DNA cleavage complexes. This entire protocol can be completed in ~2 d.

INTRODUCTION

The protocol presented here provides a practical approach for the identification of compounds that target Top1 through the induction of Top1-mediated DNA cleavage. We have successfully used this method to identify the new classes of non-camptothecin Top1 inhibitors, the indenoisoquinolines^1–6. In addition, several campto-thecin derivatives with more favorable chemical properties have been recognized using this method^7–11. A simplified flow diagram of the various stages in the identification of Top1 inhibitors in our laboratory is shown in Figure 1.

Figure 1 |.

Flow diagram to illustrate the major steps of the protocol.

Top1: background and overview

Topoisomerase I is ubiquitous and essential in mammals and flies^12,13. In yeast cells, the absence of Top1 results in nonlethal genomic instability (in particular, in their ribosomal DNA segments)¹⁴, and survival is probably due to compensation by the other topoisomerases.

The mammalian genome encodes seven topoisomerase genes: four for type I topoisomerases and three for type II topoisomerases (encoding Top2α and Top2β and Spo11). Type I topoisomerases cleave one DNA strand per catalytic cycle, whereas type II enzymes cleave both strands of the DNA duplex at one time. The four mammalian type I topoisomerase genes include, in addition to the nuclear topoisomerase I (TOP1), mitochondrial topoisomerase I (TOP1mt)^15,16 and two genes encoding topoisomerases III, one for Top3α and the other for Top3β (reviewed in refs. 17,18). Type I topoisomerases have been subdivided into two groups: type IA and IB, on the basis of the side of the DNA break to which the enzyme becomes covalently bound as it forms its catalytic tyrosyl-DNA cleavage intermediate, referred to as the cleavage complex (see Fig. 2; reviewed in detail in refs. 17–19). Top3 enzymes belong to the type IA group along with bacterial topoisomerase I as they form 5′-DNA tyrosyl adducts like the type II topoisomerases. Top1 and Top1mt belong to the type IB group and are the only known topoisomerases forming 3′-phosphotyrosyl bonds in eukaryotic cells.

Figure 2 |.

Structure and mechanism of the Top1 cleavage complex (Top 1Cc) trapping by a specific Top1 inhibitor (indenoisoquinoline derivative)^4,5,55. (a,b) Top1 nicking–closing reaction^17,18. (a) Top1 is generally bound noncovalently to DNA. The Top1 catalytic tyrosine (Y723 for human nuclear Top1) is represented in red (Y). (a to b) Top1 cleaves one strand of the duplex, as it forms a covalent phosphodiester bond between the catalytic tyrosine and the 3′-DNA terminus. The other DNA terminus is a 5′-hydroxyl (OH). (b) The Top 1Cc allows rotation of the 5′-terminus around the intact strand, which relaxes DNA supercoiling (purple dotted circle with arrowhead). (b to a) Following DNA relaxation, Top1 religates the DNA. Under normal conditions, the religation (closing) reaction rate constant is much higher than the cleavage (nicking) rate constant. More than 90% of the Top1–DNA complexes are noncovalent³⁸. (c) Top1 inhibitors (green and red polygon; structures shown in Figs. 3 and 4) trap Top 1Cc by the binding of one drug molecule at the enzyme–DNA interface between the base pairs flanking the Top1-mediated DNA cleavage site (by convention positions −1 and +1). (d,e) Lateral views of a Top 1Cc trapped by an indenoisoquinoline derivative (drug shown in green and red; DNA in dark blue; Top1 in yellow)^4,5,55. (d) Top1 is shown in a surface view to represent the depth of the indenoisoquinoline binding pocket. (e) Top1 is represented in ribbon diagram to allow visualization of the catalytic tyrosine (Y; red) and to show the drug intercalation between the −1 and +1 base pairs^4,22,55.

Topoisomerase I relaxes DNA supercoiling in the absence of an energy cofactor by nicking the DNA and allowing rotation of the broken strand around the Top1-bound DNA strand (Fig. 2b, curved arrow). Crystal structures of Top1^20–22 show the enzyme encircling the DNA tightly like a clamp (see Fig. 2d,e), which accounts for the fact that Top1 controls the processive relaxation of supercoiled DNA²³. Once the DNA is relaxed, Top1 religates the breaks by reversing its covalent binding. Under normal conditions, the cleavage intermediates are transient and religation is favored over cleavage. Top1 can only dissociate from DNA upon DNA religation (see ANTICIPATED RESULTS for more information).

DNA topoisomerases are particularly vulnerable during their cleavage intermediate step (or cleavage complex). Indeed, cleavage complexes are normally so transient that they are not detectable in spite of their frequency throughout the genome. Although Top1 cleavage complexes (Top 1Cc) are specifically and reversibly trapped by camptothecin (CPT) and other drugs that are the focus of this protocol, it is important to recognize that cellular Top 1Cc can also accumulate to high levels under two other conditions—DNA modifications^21,24–42 and apoptosis^43–51 (see Fig. 1d in ref. 19). As religation of Top 1Cc requires nucleophilic attack of the tyrosyl–DNA–phosphodiester bond by the free DNA end (the 5′-hydroxyl end; see Fig. 2b), it is critical for the 5′-hydroxyl-DNA end to be perfectly aligned with the scissile tyrosyl–phosphodiester bond. Any misalignment prohibits religation and leads to an accumulation of Top 1Cc. Hence, Top 1Cc accumulate at sites of base mismatches, base oxidation, abasic sites, carcinogenic adducts and preexisting DNA breaks (for review, see refs. 32,52) because of the misalignment of the 5′-hydroxyl end resulting from such DNA lesions. The occurrence of apoptotic Top 1Cc has been documented in a broad range of cell types and in response to various apoptotic stimuli, including staurosporine (an ubiquitous apoptosis inducer⁵³), arsenic trioxide⁴⁵, tubulin and topoisomerase II inhibitors⁴⁸, UV-C⁴⁶, tumor necrosis factor-α⁴⁷, Fas and tumor necrosis factor-related apoptosis-inducing ligand⁵¹. The apoptotic Top 1Cc are among the earlier biochemical changes observed during apoptosis. They result, at least in part, from oxidative DNA modifications generated by reactive oxygen species produced during apoptosis^44,51.

Top1 inhibitors: background and overview of current drugs

Topoisomerase I inhibitors act as interfacial inhibitors^54,55 and are commonly referred to as Top1 poisons because their antitumor activity is due to the trapping of Top 1Cc rather than to inhibition of Top1 catalytic activity^19,56,57. Hence, the following protocols will focus on the methods used to identify the DNA cleavage resulting from the trapping of Top 1Cc by Top1 inhibitors.

Figures 3 and 4 include the chemical structures of the main Top1 inhibitors currently known. CPT (Fig. 3a) was first isolated from the bark of the Chinese tree, Camptotheca acuminata⁵⁸, by the National Cancer Institute (NCI). The tree is immune to the drug because it encodes a Top1 with a point mutation Asn722Ser⁵⁹, which confers high resistance to CPT⁶⁰. CPTcarboxylate was tested clinically in the mid-1970s and showed anticancer activity, but was discontinued because of side effects⁵⁸. It is after the discovery that Top1 was the cellular target of CPT⁶¹ that the water-soluble derivatives of CPT—topotecan and irinotecan (CPT-11)—were developed¹⁹. Newer camptothecin derivatives are in clinical trials⁶². Gimatecan (LBQ 7707) is being developed as an oral formulation by Novartis⁶³.

Figure 3 |.

Structures of camptothecin derivatives. (a) The active camptothecin alkaloid is the 20-(S) enantiomer (as shown with the 20-hydroxyl above the plane). Camptothecin is spontaneously and reversibly converted to its carboxylate derivative, which binds serum albumin and is inactive against Top1. (b) The two camptothecins approved for cancer chemotherapy are topotecan and irinotecan. Both are water soluble, but irinotecan is a prodrug and needs to be converted to SN-38. (c) Camptothecin derivatives in clinical trials. Gimatecan can be given orally. (d) Two strategies have been used to overcome the inactivation of camptothecins by E-ring opening. An additional methylene group yields homocamptothecins with a more stable E-ring^9,68. However, once converted to carboxylates, homocamptothecins cannot reverse to the active E-ring-closed form. Removal of the oxygen atom in the E-ring yields keto derivatives that cannot undergo E-ring opening⁷⁰. S39625 has just begun clinical trials¹¹. Substitutions on the camptothecin structure are shown in red.

Figure 4 |.

Structures of the three chemical families of noncamptothecin Top1 inhibitors. The indolocarbazole derivative⁹⁷, edotecarin, is in Phase II clinical trials. Two indenoisoquinolines are in preclinical development^3,6. ARC-111 (topovale) (or one of its derivatives) is also in preclinical development^76,98.

Camptothecins have several limitations^19,62. First, their α-hydroxylactone E-ring is readily converted into a carboxylate (Fig. 3a), which is only weakly active against Top1^64,65 and also binds tightly to serum albumin⁶⁶. Second, Top 1Cc need to be maintained long enough to be converted into DNA damage⁶⁷. Indeed, camptothecins rapidly diffuse from the Top 1Cc, which means that they must be given as prolonged infusion to maintain persistent cleavage complexes. Third, cells that overexpress drug efflux pumps are resistant to camptothecins. And fourth, camptothecins produce side effects (such as leucopenia and diarrhea) that limit the doses that can be safely administered and, therefore, antitumor efficacy. A summary of the rationale for developing noncamptothecin Top1 inhibitors is provided in Table 1.

TABLE 1 |.

Rationale for developing noncamptothecin Top1 inhibitors.

1. Camptothecins are among the most effective anticancer drugs recently introduced in cancer chemotherapy. Therefore, Top1 is a validated target for cancer treatment.

2. Drugs with common targets are known to have different pharmacological and anticancer activity (e.g., Top2 poisons or tubulin inhibitors).

3. Camptothecins have limitations:

(a) inactivation by E-ring opening to carboxylate derivatives;

(b) rapid dissociation from Top1 cleavage complex;

(c) Substrates for multidrug resistance transporters;

(d) Narrow therapeutic index with toxicity to bone marrow and intestine.

Two modifications of the camptothecin E-ring have been introduced to alleviate the drug inactivation by E-ring opening (Fig. 3d). Addition of a methylene group, as in diflomotecan (BN80915), stabilizes the E-ring, although retaining Top1 inhibition⁶⁸. Diflomotecan is in early clinical trials⁶⁹. A more efficient way to stabilize the E-ring is to remove the lactone group, which completely blocks E-ring opening as in the keto derivatives (Fig. 3d)⁷⁰. The cyclobutane methylenedioxy derivative¹¹ developed by Servier, S39625, is expected to start clinical trials this year.

Noncamptothecin Top1 inhibitors were searched for immediately after discovering that Top1 was the cellular target of camp-tothecins. Screening of chemical libraries and natural products with purified Top1 and isolated DNA substrates led to the discovery of diverse Top1 inhibitors (for review, see refs. 19,62,71,72), including the indolocarbazole and phenanthroline derivatives. Simultaneously, computer analyses of drug activity profiles in the NCI Developmental Therapeutics Program led to the discovery of the first indenoisoquinoline using comparative cytotoxicity pattern profiles across the 60 cell lines of the NCI anticancer screen¹. This approach is commonly referred to as the COMPARE algorithm⁷³.

Indolocarbazoles⁷⁴ are the most advanced noncamptothecin Top1 inhibitors in clinical development, with edotecarin in phase II clinical trials (Fig. 4)⁶². Several other indolocarbazoles are also undergoing clinical trials in Japan⁷⁵. Nitidine and phenanthridine derivatives were developed in the mid-1990s. Topovale (ARC-111) is the lead phenanthridine for clinical development (Fig. 4)^62,76. Among more than 400 indenoisoquinolines evaluated as Top1 inhibitors⁷⁷, three derivatives have been selected for preclinical development: MJ-III-65 (NSC 706744), NSC 725776 and NSC 724998 (see refs. 6,78). The indenoisoquinolines have several differential characteristics (and advantages) compared with the camptothecins. First, they are chemically stable, as they do not contain the labile hydroxylactone function of camptothecins. Second, they trap Top 1Cc at different DNA sequences compared with the camptothecins, and therefore target the genome at different sites^1,3,6,78, which might translate into different cellular effects and clinical activity profile. Third, the indenoisoquinolines overcome drug efflux-mediated resistance⁶. And fourth, the Top 1Cc induced by indenoisoquinolines in biochemical systems and in cells are markedly less reversible than those induced by camptothecins^3,6,78. Hence, the pharmacokinetics of indenoisoquinolines should allow the formation of persistent Top 1Cc, potentially enabling shorter infusion times.

The step-by-step protocol presented here has been used successfully to identify a number of compounds with the propensity to stabilize Top1–DNA covalent complexes^{1,3,4,77,79–81}. The limitations of this protocol are essentially related to the availability of the inhibitors (i.e., the chemistry). Nevertheless, camptothecin is available commercially and can serve as reference.

Principle of the Top1-mediated DNA cleavage assay

As stated above, the hallmark of Top1 inhibitors is the DNA cleavage event mediated by the formation of the cleavage complex in which the enzyme is covalently attached to the 3′-end of the cleaved DNA. Such Top1-dependent DNA cleavage can be conveniently monitored using the DNA cleavage assay described herein. In this assay, the enzyme and DNA are mixed and allowed to establish a cleavage/religation equilibrium. In the absence of denaturing agents or specific Top1 inhibitors, Top1 is kinetically competent and capable of mediating DNA religation. Indeed, the equilibrium between the two opposing transesterification reactions greatly favors the religated state (Fig. 2a), such that the fraction of the enzyme that is covalently bound to the DNA is virtually undetectable (Fig. 2b). However, Top1 inhibitors, such as those shown in Figures 3 and 4, have the propensity to selectively bind at the interface of the Top1–DNA covalent complex^4,5,22,54,55 and consequently impede the rate of religation (Fig. 2c). Exposure of these drug-stabilized cleavage complexes to a strong protein denaturant, such as sodium dodecyl sulfate (SDS), leads to the rapid denaturation of Top1 covalently bonded to nicked DNA. Following DNA duplex denaturation and electrophoretic resolution on a polyacrylamide gel, cleavage products are represented by the appearance of discrete low molecular weight species (Fig. 5).

Figure 5 |.

Cartoon illustrating the advantages of using a 3′-end-labeled DNA substrate. (a) Example of theoretical products that are produced following the Top1-mediated DNA cleavage reaction in the presence of a Top1 inhibitor. Blue circles correspond to the 3′-end label. (b) Migration of the denatured DNA fragments on a denaturing polyacrylamide gel. Labeled DNA fragments, which are visible after the PhosphorImager scan, are shown in black. Unlabeled DNA fragments are shown in gray.

Principle of the Top1–DNA cleavage complex reversal assays

Similar to CPT, most Top1 inhibitors bind reversibly to the Top1 cleavage complex and act primarily by blocking DNA religation (Fig. 2c). The enhanced steady-state level of Top1 cleavage complexes reflects a new equilibrium between DNA cleavage and religation (slowed down by the inhibitor). To establish unambiguously whether an agent acts in this manner rather than by inducing the formation of Top1 cleavage complexes, it is important to perform reversal assays^6,7,11,65,78. The principle of this assay is derived from the findings that high salt conditions (0.35 M NaCl) or heat (65 °C) has the propensity to inhibit the formation of new Top1 cleavage complexes by interfering with de novo binding of the enzyme to DNA, whereas having no effect on the DNA religation activity of Top1^7,65. Thus, by preventing the forward reaction of Top1 cleavage complex formation, the Top1 religation rate may be examined³⁸.

For the reversal assay, Top1–DNA cleavage complexes are allowed to equilibrate in the presence of an inhibitor. These complexes are then subjected to a high salt or heat treatment, which blocks the formation of new cleavage complexes, although permitting DNA religation. Reversal is generally fast with a half-life for CPT within 2 min at 37 °C (ref. 7). To better quantify reversal (religation) kinetics, we recommend performing salt reversal at low temperature by cooling down the reactions to 10 or 0 °C immediately before adding salt^11,38,78. The amount of DNA strand cleavage remaining after ‘reversal’ treatment is monitored over time, and forward and reversal rates can be readily computed³⁸. Camptothecin is a convenient positive control, as its inhibitory effect on religation kinetics is well established^7,38,61,78.

Experimental design

DNA substrate.

The DNA cleavage assay begins with the design of an optimal DNA substrate. Earlier studies determined that the minimum DNA duplex region required for a Top1-mediated reaction in vitro is a sequence containing nine nucleotides on the scissile strand and five nucleotides on the noncleaved strand⁸². Since then, several unique oligonucleotide sequences have been designed and exploited that contain a single high-efficiency Top1 cleavage site^3,82–86. Although these short synthetic oligonucleotide DNA substrates are easily manipulated and are an advantageous tool to study the kinetics of the Top1 reaction in vitro, they are limited by the quantity, and sometimes quality, of the Top1 cleavage sites. In contrast, larger DNA fragments (>50 bp in length) can be used to increase the number of Top1 cleavage sites and thus permit analysis of the DNA sequence specificity of the Top1 cleavage sites in the presence of an inhibitor. However, quantifying the end results can be difficult. In practice, we have found it convenient to initially investigate the ability of a prospective Top1 inhibitor to induce Top1-mediated DNA cleavage using a large DNA fragment. Conventional oligonucleotide DNA substrates are then employed to further characterize the drug activities. For the purposes of the protocols described here, we have restricted the DNA substrate to a 181-bp fragment from the pBluescript SK(−) phagemid DNA^3,86; however, because of the high frequency of Top1 sites in most DNA sequences⁸⁵, almost any DNA fragment may be used irrespective of its origin.

End-labeling of DNA substrate.

Regardless of DNA substrate choice (PCR-generated DNA fragments or oligonucleotides), it is highly recommended to label the DNA substrate at the 3′-end. In the Top1 cleavage complex, the enzyme links to the 3′-terminus of the cleaved strand (see Figs. 2b and 5a) and thus labeling of the 3′-end confines analysis to the cleavage products that are not covalently linked to the Top1. Unless complete proteolytic digestion of Top1 is carried out on a 5′-end-labeled cleavage product before electrophoresis, a substantial amount of the label will remain in or near the well of the gel (see Fig. 5b). 3′-End-labeled DNA substrates also allow for unambiguous mapping of the DNA cleavage sites, whereas 5′-end-labeled fragments do not because polypeptide residues remain covalently bound to the 3′-end tyrosyl even after extensive digestion. Another advantage of 3′-labeling is that the ³²P-radioisotope is incorporated inside the DNA backbone, which allows the use of nuclear extracts. Phosphatases, which are generally present in nuclear extracts, readily remove 5′-³²P-label. There are two reasons for using nuclear extracts. First, nuclear extracts are easier and cheaper to prepare than purified Top1. Second, nuclear extracts allow comparative studies between cell types or cells under various conditions.

Choice of topoisomerase source.

There is no limitation to the source of the Top1 used in the protocols described here. For example, sources of Top1 have ranged from nuclear extracts to purified preparations of recombinant enzyme. This can be extended to nuclear extracts from both drug-resistant and sensitive cell lines as well as wild-type and mutant recombinant enzymes¹¹. An optional protocol for the preparation of nuclear extracts is described (see Steps 13–24). Additional methods for the overexpression and purification of various forms of Top1 have been described previously (see ref. 87).

Controls and DNA markers.

The following are important control samples that should be prepared and processed in parallel with the experimental samples.

1. The negative enzyme controls include all the reaction constituents with the exception of the enzyme. As an alternative, the enzyme source may also be heat-denatured and added to the negative enzyme control. In general, these controls ensure the ‘cleanliness’ of the substrate DNA and the lack of effect of the drug on the DNA substrate in the absence of Top1. The control samples should exhibit a single, intense band, which electrophoreses with a mobility corresponding to the full-length DNA. The appearance of shorter DNA fragments may be indicative of DNA degradation or a chemical effect of the drug on DNA.

2. The negative inhibitor control includes the DNA substrate and the enzyme source minus any Top1 inhibitor. This control provides a measure of the background Top1 cleavage of the DNA substrate. Typically, low to undetectable DNA cleavage is observed.

3. The positive inhibitor control includes a known Top1 inhibitor, such as CPT, for verification of the assay performance characteristics. In the presence of CPT, a depletion of the full-length DNA and the emergence of shorter DNA fragments should be observed.

4. A Maxam–Gilbert sequencing reaction is performed to aid in the precise mapping of the Top1 Cleavage sites (see Steps40–48). It is important to recognize that Maxam–Gilbert sequencing reactions yield DNA cleavage fragments that result from elimination of the chemically modified base (generating a 3′-phosphate). Thus, the resulting fragments migrate slower (0.5–1 base) than the corresponding Top1-generated fragments.

5. 3′-End-labeled oligonucleotide size markers can be run alongside the Top1–drug and Maxam–Gilbert reaction lanes to unambiguously determine the position of the cleavage site(s). It is recommended to use oligonucleotide size markers with similar sequence composition as the predicted cleavage products (inferred from the Maxam–Gilbert markers), as oligonucleotides with different sequences can exhibit different electrophoretic mobilities.

Alternative methods.

One of the most widely used alternatives to the DNA cleavage assay in labeled DNA fragments is the DNA relaxation assay^61,88,89. This assay is based on the transformation of a supercoiled plasmid DNA substrate into a population of relaxed products by the catalytic activity of Top1. Although similar in sequence and molecular weight, the compact nature of the supercoiled DNA and its relaxed topo-isomers are easily distinguished using agarose gel electrophoresis. One advantage to the DNA relaxation assay is its high sensitivity (i.e., low stoichiometry). In addition, this assay can be easily performed using a commercially available kit (Topogen). However, as the DNA relaxation assay contains all the steps of the Top1 reaction—binding, cleavage, strand transport, religation and enzyme turnover—it is difficult to differentiate the influence of drugs on the individual steps of Top1 action. In contrast, the DNA cleavage assay allows for direct analysis of the cleavage step of the Top1 reaction because the processivity is irrelevant and cleavage is stoichiometric with enzyme. The DNA relaxation assay is also very sensitive to DNA intercalating drugs that counteract Top1 activity as they unwind DNA⁸⁸. Thus, even if a drug inhibits Top1-mediated DNA relaxation, it is necessary to perform DNA cleavage assays to differentiate between Top1 poisons (which trap Top 1Cc), DNA intercalators (which unwind the DNA without directly affecting Top1) and Top1 catalytic inhibitors (which inhibit Top1 catalytic activity directly but without trapping Top 1Cc).

DNA cleavage assays can also be performed as a modification of the DNA relaxation assay. Indeed, nicked circular DNA (sometimes referred to as ‘form II’) can be separated from relaxed circular DNA (‘form II_O’) and supercoiled substrate DNA (‘form I’) by adding an intercalating agent (ethidium bromide (EtBr) or chloroquine) in the agarose gel electrophoresis buffer^61,89,90. Binding of the intercalating drug generates positive supercoiling in the closed DNA, which then increases its electrophoretic mobility⁸⁸. In contrast, intercalators cannot overwind nicked DNA as the breaks provide swivel points. Cleavage assays with supercoiled DNA require much higher amounts of Top1 enzyme than the relaxation assay⁸⁹. It is much less sensitive than the cleavage assays described in this protocol, as there is always a background level of nicked DNA in plasmid or viral DNA duplex circles. Moreover, finding the right concentration of intercalators to resolve the nicked and relaxed circular DNA is often delicate. Finally, chloroquine gels are difficult to stain with EtBr, and contaminating nucleases often increase the background levels of nicked DNA, which can obscure the drug-induced Top 1Cc.

Cellular assays will not be reported in this protocol. Commonly used techniques included alkaline elution, which led to the discovery of topoisomerase inhibitors^6,91, SDS-KCl precipitation assays92,93 and ICE-bioassays6,36,43.

MATERIALS

REAGENTS

• pBluescript SK(−) Phagemid Vector (Stratagene, cat. no. 212206)

• PCR Primers, pSK-forward: 5′-GCCTCTTCGCTATTACGCCAG-3′; pSK-reverse: 5′-CGAACTATAGCTTAAGGACGTCGG-3′ (Integrated DNA Technologies)

• Deoxynucleotide set (100 mM dNTPs; New England BioLabs, cat. no. N0446S)

• Taq DNA polymerase (5 U μl⁻¹), including 10× Taq buffer and 25 mM MgCl₂ (Fermentas, cat. no. EP0071)

• QIAquick Gel Extraction Kit (Qiagen, cat. no. 28704)

• HindIII (New England BioLabs, cat. no. R0104)

• Agarose (Sigma, cat. no. A9539)

• EtBr solution (Sigma, cat. no. E1510) Ethidium bromide is mutagenic. Always use gloves when handling gels and solutions containing ethidium bromide.

• 10× Tris-borate-ethylenediaminetetraacetic acid (TBE; Invitrogen, cat. no. 15581–028)

• [α-³²P] dGTP (PerkinElmer, cat. no. BLU014Z) Use appropriate safety precautions for radioactive material handling, use, storage and disposal.

• Klenow fragment (New England BioLabs, cat. no. M0212)

• Mini Quick Spin DNA Columns (Roche, cat. no. 1814419001)

• Dimethyl sulfoxide (DMSO; Sigma, cat. no. D5879)

• CPT (Sigma, cat. no. C9911)

• Monopotassium phosphate (KH₂PO₄; Sigma, cat. no. P8709)

• Dipotassium phosphate (K₂HPO₄; Sigma, cat. no. P8584)

• Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA; Sigma, cat. no. 03777)

• Triton X-100 (Sigma, cat. no. T8787)

• Glycerol (Invitrogen, cat. no. 15514–011)

• β-Mercaptoethanol (Sigma, cat. no. M7154)

• Complete mini protease inhibitor cocktail tablets (Roche, cat. no. 11836153001)

• Tris-HCl, pH 7.5 (Quality Biological Inc., cat. no. 351–006-101)

• Potassium chloride (KCl; Sigma, cat. no. 60142)

• Magnesium chloride (MgCl₂; Sigma, cat. no. 63030)

• EDTA, pH 8.0 (Sigma, cat. no. 03690)

• Formic acid (Sigma, cat. no. F0507)

• Sodium acetate (Sigma, cat. no. 71196)

• Yeast transfer RNA (tRNA; Sigma, cat. no. R5636)

• Piperidine (Sigma; cat. no. 571261)

• Ethanol (Sigma; cat. no. E7148)

• BSA (Sigma, cat. no. B8667)

• DTT (Sigma, cat. no. 43816)

• Sodium chloride (NaCl; Sigma, cat. no. S5150)

• Top1 can be directly expressed from a variety of expression systems, cell lines, or tissues. Commercial sources are also available (Topogen, cat. no. 2005H-RC1; Sigma, cat. no. T9069; GenWay, cat. no. 10–663-45329; Jena Bioscience, cat. no. PR-735). All such sources are compatible with this assay

• SDS (Quality Biological Inc., cat. no. 351–032-101)

• Formamide (Sigma, cat. no. F7503)

• Xylene cyanol (Sigma, cat. no. X4126)

• Bromophenol blue (Sigma, cat. no. B8026)

• AccuGel, 40% (wt/vol) acrylamide:bisacrylamide (19:1) (National Diagnostics, cat. no. EC-850). Ready-to-use solutions of acrylamide/bisacrylamide are commercially available so as to avoid the handling of harmful acrylamide powder. Unpolymerized acrylamide and bisacrylamide are neurotoxins and suspected carcinogens and should be handled accordingly.

• Urea (Sigma, cat. no. U1250)

• Ammonium persulfate (APS; Sigma, cat. no. A3678)

• N,N,N′,N′-tetramethylethylenediamine (TEMED; Sigma, cat. no. T9281)

EQUIPMENT

• Thermal cycler

• Agarose gel electrophoresis equipment

• Vertical/sequencing electrophoresis equipment, including notched glass plates (33 cm × 42 cm), spacers (0.4 mm) and combs

• UV transiluminator for visualization of EtBr-stained nucleic acids

• Heat block

• Scintillation counter

• Speed Vac

• Power supply for electrophoresis

• Equipment to work with ³²P (i.e., Plexiglas shielding and Geiger counter)

• Whatman 3 MM paper

• Gel drying system

• PhosphorImager scanner and storage PhosphorImager cassette/screen

• ImageQuant software (Molecular Dynamics)

REAGENT SETUP

10 mM dNTP mix for PCR

Individual dNTPs (dATP, dCTP, dGTP and dTTP) are combined and diluted to obtain a mixture of each dNTP at 10 mM.

0.5 mM dNTP mix minus dGTP

Individual dNTPs (dATP, dCTP and dTTP) are combined and diluted to obtain a mixture of each dNTP at 0.5 mM. 103 Top1 reaction buffer 100 mM Tris-HCl (pH 7.5), 500 mM KCl, 50 mM

10× Top1 reaction buffer

100 mM Tris-HCl (pH 7.5), 500 mM KCl, 50 mM MgCl₂, 1 mM EDTA and 150 μg ml⁻¹ BSA. Store aliquots at −20 °C.

Human topoisomerase I

We routinely express and purify recombinant human Top1 from insect cells using a baculovirus construct for the full-length human Top1 cDNA⁹⁴.

10× nuclear extract buffer (optional)

1.5 M NaCl, 10 mM KH₂PO₄ (pH 6.4), 50 mM MgCl₂ and 10 mM EGTA.

10% (wt/vol) APS solution

Dissolve 1 g of APS in 10 ml of water and store at 4 °C. Replace the stock solution every 2–3 weeks.

16% (wt/vol) polyacrylamide gel stock solution

Combine the following components in a 1-liter beaker or bottle.

Component	Volume/amount	Final amount/concentration
40% (wt/vol) acrylamide:bisacrylamide (19:1) solution	400 ml	16%
10× TBE	100 ml	1×
Urea	420.42 g	7 M
ddH2O	To 1 liter	—

Gel solution should be stored at room temperature (20–25 °C) in a dark bottle or clear bottle wrapped with aluminum foil to prevent deamination.

Preparation of a 16% (wt/vol) polyacrylamide gel

Preparing the glass plates.

Clean the surface of the glass plates. We routinely clean the glass plates with detergent and then twice with a commercial glass cleaner, such as Windex. Other chemicals, such as ethanol or acetone, may also be used. In addition, the interior surface of the plates can also be coated with a siliconizing fluid (e.g., Sigmacote), which permits easy removal of the gel from the glass plates after electrophoresis. It is important to clean glass plates thoroughly to prevent the formation of bubbles in the gel.

Assembling the glass plates.

Place the side spacers (0.4 mm) between the glass plates and secure the plates together with binding clamps (sequencing tape or a casting boot may be substituted for clamps). Check that the bottom of both glass plates and spacers are flush with one another to prevent leaking.

Pouring the gel.

Transfer 50–60 ml of the 16% (wt/vol) polyacrylamide solution, prepared as described in REAGENT SETUP, into a clean beaker. To this solution, add 300 μl of 10% (wt/vol) APS and 30 μl of TEMED. Swirl gently and then immediately and carefully pour the solution into the assembled glass plate setup. Insert the selected gel comb and allow the gel to polymerize for a minimum of 30 min. Any remaining gel solution can be used as a polymerization control. TEMED and APS should be added just before pouring the gel. Polymerized gels can be stored for several hours at room temperature.

Denaturing formamide dye

80% (vol/vol) formamide, 10 mM NaOH, 1 mM EDTA, 0.25% (wt/vol) bromophenol blue and 0.25% (wt/vol) xylene cyanol.

PROCEDURE

Preparation of the DNA substrate 24 h

• 1
  The DNA substrate is generated from the pBluescript SK(−) phagemid vector by PCR using pSK-forward and pSK-reverse primers (see REAGENTS). Set up the PCR in a 200-μl thin-walled PCR tube as follows:

  Component	Volume	Final amount/concentration
  10× Taq polymerase buffer	5.0 μl	1×
  MgCl2 (25 mM)	3.0 μl	1.5 mM
  dNTPs mix (10 mM)	1.0 μl	0.2 mM
  pSK-Forward (10 μM)	1.5 μl	0.3 μM
  pSK-Reverse (10 μM)	1.5 μl	0.3 μM
  pBluescript SK(—) (0.5 μg μl−1)	0.5 μl	0.25 μg
  Taq DNA polymerase (5 U μl−1)	1.0 μl	5 U
  ddH2O	36.5 μl	—
  Final	50 μl

  It is convenient to prepare multiples of the above PCR to avoid repeating Steps 1–7.

• 2
  Amplify the target sequence using the following PCR cycling conditions tabulated below.

  Cycle	Denaturation	Annealing	Extension
  1	95 °C for 2 min	—	—
  2–25	94 °C for 10 s	60 °C for 30 s	72 °C for 1 min
  26	—	—	72 °C for 5 min

  Samples can be held at 4 °C in the thermal cycler overnight.

• 3Check the quality of the PCR product by running a small aliquot (5 μl) of the PCR on a 1% (wt/vol) agarose gel containing 0.5 μg ml⁻¹ ethidium bromide. If a single band of the expected size (202 bp) is observed, proceed to Step 4.
• 4Run a 1% (wt/vol) agarose gel containing 0.5 μg ml⁻¹ ethidium bromide with an adequately sized well to load the entire volume of the pooled PCRs.
• 5Visualize the gel by UV illumination and excise the band that corresponds in size to the 202-bp product with a clean, sharp scalpel or razor blade. Place the gel fragments into separate 1.5-ml microcentrifuge tubes. Remove as much excess agarose as possible.

  Minimize DNA exposure to UV light to prevent photochemical damage.

• 6Recover the agarose-encased DNA using an appropriate DNA extraction method. We routinely use the QIAquick gel extraction kit according to the manufacturer’s instructions.
• 7Check the DNA purification yield by UV spectrophotometry at 260 nm and/or quantitative analysis on an agarose gel. Aliquot and store the purified DNA at −20 °C.
• 8Digest an aliquot of the confirmed PCR product with HindIII restriction endonuclease. To achieve complete digestion, we use a 40-μl reaction product containing 0.5–1 μg of DNA and 20 U of restriction enzyme and incubate for 15 h at 37 °C. We routinely supplement the digestion reaction with an additional 10 U of restriction enzyme of the final hour of incubate.

  An agarose gel may be run to check that complete digestion has occurred (product size ~181 bp).

  Follow all standard radioactivity safety procedures for the remainder of this protocol. Consult your institution safety office for information regarding safe handling of radioactive material.

• 9
  Following digestion, the HindIII restriction site 5′-overhangs are radiolabeled by fill-in reaction with the Klenow fragment of DNA polymerase and [α-³²P] dGTP in the presence of unlabeled dCTP, dATP and dTTP. The reaction is set up as follows:

  Component	Volume	Final amount/concentration
  HindIII-digested DNA	40 μl	0.5–1 μg
  10× Klenow buffer	6 μl	1×
  dCTP, dATP and dTTP mix (0.5 mM)	3 μl	25 μM
  [α-32P] dGTP (20 μCi μl−1)	5 μl	100 μCi
  ddH2O	5 μl	—
  Exo-free Klenow fragment (5 U μl−1)	1 μl	5 U
  Final	60 μl	—

  Use fresh [α-³²P] dGTP for optimal incorporation.

• 10Incubate the reaction for 15 min at 25 °C and terminate by the addition of EDTA (pH 8.0) to a final concentration of 10 mM or by heat inactivation for 5–10 min at 70 °C.
• 11Remove the unincorporated radiolabeled nucleotides from the 3′-end-labeled DNA using a mini Quick Spin DNA Column (Sephadex G-50) according to the manufacturer’s instructions.
• 12Measure the specific activity of the labeled DNA by measuring 1 μl in a scintillation counter. An activity ≥100,000 cpm μl⁻¹ will provide the best results.

  Radiolabeled DNA can be stored for weeks at 4 °C; however, it is advised to carry out the experiments promptly due to the short half-life (14.3 days) of the ³²P radioisotope.

Preparation of nuclear extracts (optional) 2 h

• 13Prepare fresh 1× nuclear extract buffer and 1× high-salt nuclear extract buffer using the following measurements in the tables below. Commercial nuclear extraction kits are also available, if desired.
  1× nuclear extract buffer

  Components	Volume per sample	Final amount/concentration
  10× nuclear extract buffer (see REAGENTS SETUP)	20 ml	1×
  Glycerol (100%)	20 ml	10%
  β-Mercaptoethanol (14.2 M)	140 μl	10 mM
  Protease inhibitor tablets	6 tablets	—
  ddH2O	To final volume of 200 ml	—

  1 high-salt nuclear extract buffer

  Components	Volume per sample	Final amount/concentration
  10× nuclear extract buffer (see REAGENTS SETUP)	1 ml	1×
  NaCl (5 M)	0.7 ml	0.35 M
  Glycerol (100%)	1 ml	10%
  2-Mercaptoethanol (14.2 M)	7 μl	10 mM
  Protease inhibitor tablets	1 tablets	—
  ddH2O	To final volume of 10 ml	—

• 14Remove cell culture media and wash cells twice with an appropriate volume of ice-cold 1× PBS. For each wash, gently rock the flask for 1 min before removing the PBS.

  Typically, we harvest a minimum of 1 × 10⁷ cells. This amount of cells can be obtained from a T75 culture flask with 80–90% confluency depending upon the cell type.

• 15Trypsinize or scrape cells (with a sterile cell scraper) to remove them from the flask.
• 16Transfer cell suspension to an appropriate-sized centrifuge tube and pellet at 200g for 5 min at 4 °C. Remove the supernatant.
• 17Wash the cell pellet by resuspending in 10 ml of 1× nuclear extract buffer and centrifugation at 200g for 5 min at 4 °C. Remove the supernatant. Repeat this step twice.
• 18Lyse the cells by resuspending the cell pellet in 10 ml of 1× nuclear extract buffer containing 0.3% (vol/vol) Triton X-100 (30 μl). Rotate gently for 10 min at 4 °C.
• 19Collect the cell nuclei by centrifugation at 350g for 10 min at 4 °C. Carefully remove the supernatant as much as possible and avoid disrupting the pellet. This supernatant contains the cytoplasmic extracts, which can be stored at −80 °C, if desired.
• 20Wash the nuclei by resuspending in 1 ml of 1× nuclear extract buffer and centrifugation at 350g for 5 min at 4 °C. Repeat this step twice.
• 21Completely resuspend the nuclear pellet in 300 μl of 1× high-salt nuclear extract buffer by gently pipetting up and down.
• 22Gently rotate the nuclear mixture for 30 min at 4 °C.
• 23Remove insoluble nuclear components by centrifugation at 12,000g for 10 min at 4 °C.
• 24Collect supernatant (nuclear extract) and quantify amount of protein using standard protein estimation assay.

  Prepare aliquots of the nuclear extract to prevent multiple freezing and thawing.

Top1-mediated DNA cleavage assay 1 h

• 25Dissolve CPT or other putative Top1 inhibitors in DMSO to a final concentration of 10 mM. Make four serial tenfold dilutions in DMSO to test a broad range of concentrations (final concentrations from 0.1 to 100 μM).
• 26Prepare a master mix for the required number of reactions plus the appropriate controls (see Experimental design) using the following measurements in the table below.
  Control reactions typically consist of a negative enzyme control and a negative inhibitor control. Alternatively, a heat-denatured recombinant Top1 or nuclear extract may be substituted as a ‘no enzyme’ control to correct for any nonspecific DNA interaction. CPT is typically tested at one or more concentrations as a positive control.

  Components	Volume per sample	Final amount/concentration
  3′-End-labeled DNA	1 μl	~100,000 c.p.m.
  10× Top1 reaction buffer (see REAGENT SETUP)	2 μl	1×
  DTT (2 mM)	2 μl	0.2 mM
  ddH2O	11 μl	—

  When needed, adjust the volume of DNA to have at least 100,000 c.p.m. per reaction. Correct the volume of ddH₂O accordingly.

  Always make 10% more master mix than needed to compensate for pipetting error.

• 27Transfer 2 μl of the serial diluted 10× drug stocks to separate microcentrifuge tubes. Add 2 μl of DMSO to the control reactions.
• 28Add 2 μl of Top1 (50–75 U) or nuclear extract (see Steps 13–24) to each reaction tube. Add 2 ml of 1× reaction buffer to the negative enzyme control.

  One unit of Top1 is equal to the amount of enzyme able to catalyze the relaxation of 0.3 μg of supercoiled plasmid DNA under standard conditions. Commercial sources of recombinant Top1 are normally expressed in U μl⁻¹ and thus volumes can be derived from the concentration provided. The amounts of Top1 obtained from other sources, such as direct purification from expression systems or from cell lines (i.e., nuclear extracts), should be optimized for this assay due to variability from one preparation to another. We routinely optimize the Top1 reaction by assaying serial dilutions of the Top1 source in the presence of CPT. In general, ~50 ng of recombinant Top1 and a range of 0.5–2 μg of total protein from nuclear extracts are required per reaction. It is best to start with the highest possible concentration of Top1 or nuclear extract, and dilute Top1 or nuclear extract as needed if the substrate DNA is completely cleaved during the Top1 reactions.

• 29Initiate the reaction by adding 16 μl of master mix to each sample for a final volume of 20 μl.
• 30Mix gently and incubate for 20 min at 25 °C.

  The rate of the Top1 reaction can be altered by adjusting the temperature of the reaction. In addition, time course experiments may be performed to investigate the forward kinetics of Top 1Cc formation for a given inhibitor.

• 31To terminate the reactions and trap the Top 1Cc, add 0.5% (wt/vol) SDS (final concentration), followed by the addition of two volumes of denaturing formamide dye (see REAGENT SETUP).

  After the addition of denaturing formamide dye, samples can be stored at room temperature for several days.

• 32Follow Steps 49–57 for denaturing polyacrylamide gel electrophoresis (PAGE) analysis.

Top1–DNA cleavage complex reversal assay (optional) 1 h

• 33Positive hits from the Top1-mediated DNA cleavage assay can be characterized further using a cleavage reversal assay to determine the reversibility of the Top1–DNA complexes over time. For this assay, select a concentration of compound that decreases the amount of full-length DNA substrate by roughly 50% in the cleavage assay. Choose a concentration of CPT (positive control) that produces a similar decrease for comparison.
• 34
  Prepare a master mix for the required number of reactions (i.e., number of reversal time intervals) using the following measurements in the table below.

  Components	Volume per sample	Final amount/concentration
  3′-end-labeled DNA	1 μl	~100,000 c.p.m.
  10× drug concentration	2 μl	1×
  10× Top1 reaction buffer (see REAGENT SETUP)	2 μl	1×
  DTT (2 mM)	2 μl	0.2 mM
  ddH2O	11 μl	—

  Create a master mix for each drug under investigation.

  Negative enzyme and negative inhibitor control reactions are set up separately.

• 35Initiate the cleavage reaction by adding 2 μl (× the number of reactions) of Top1 (50–75 U) directly to the each master mix and incubate for 20 min at 25 °C.
• 36Initiate the reversal by the addition of NaCl to a final concentration of 0.35 M or by heating to 65 °C.
• 37Terminate the reactions at each time interval by adding 0.5% SDS (final concentration).

  Remember to remove an aliquot for the zero time point preceding application of the reversal method.

  It is convenient to set up individual microcentrifuge tubes that contain the appropriate amount of SDS before application of the reversal method to facilitate ‘on time’ termination at each reversal time interval.

• 38Add two volumes of denaturing formamide dye (see REAGENT SETUP) to each sample.
• 39Follow Steps 49–57 for denaturing PAGE analysis.

Maxam–Gilbert sequencing 2–3 h

• 40Direct mapping of the Top1 cleavage sites is achieved by sequencing the DNA molecule of interest according to the method of Maxam and Gilbert⁹⁵. A brief protocol of the Maxam–Gilbert chemical sequencing purine-specific (AG) reaction is presented here. The first step is to chemically modify the 3′-end-labeled DNA substrate by combining 20 μl (~500,000 c.p.m.) of 3′-end-labeled DNA substrate with 50 μl of formic acid. Mix and incubate for 5 min at 25 °C.

  The high amount of DNA substrate is used for two reasons. First, the final yield of this method is typically 30–50% of the starting c.p.m. Second, this will give enough sequenced DNA for multiple gels.

  Optimum incubation times may vary depending on the length of the DNA substrate.

• 41Add 180 μl of stop buffer containing 0.3 M sodium acetate, 0.1 mM EDTA and 25 μg ml⁻¹ tRNA (see REAGENTS).
• 42Precipitate the DNA by adding 750 μl of 100% ethanol and incubating at −80 °C for 30 min (or at −20 °C overnight).
• 43Pellet the DNA by centrifugation at 12,000g for 20 min at 4 °C and carefully decant the supernatant.
• 44Rinse the resulting pellet by adding 1 ml of 70% ethanol, and centrifuge at 12,000g for 5 min at 4 °C. Carefully decant the supernatant and air-dry (or drying in a Speed Vac).
• 45Catalyze the strand-scission reaction by redissolving the pellet in 100 μl of freshly diluted 1 M piperidine and heating to 90 °C for 30 min.

  Screw cap tubes or tephlon tape is recommended to ensure that the tube(s) are completely sealed to prevent changes in the piperidine concentration.

• 46Evaporate the piperidine to dryness in a Speed Vac.
• 47Resuspend the pellet in 50 μl of ddH₂O and dry in Speed Vac. Repeat 2–3 times.

  These steps are required to remove all residual piperidine. Failure to completely remove piperidine will result in smearing of bands during electrophoresis.

• 48Resuspend the pellet in the appropriate amount of denaturing formamide dye (see REAGENT SETUP) on the basis of c.p.m. remaining.

Denaturing PAGE 4–5 h

• 49Prepare a 16% denaturing polyacrylamide gel, as detailed in REAGENT SETUP. After polymerization, remove the comb carefully to maintain intact wells. Assemble the gel into the vertical electrophoresis apparatus and add 1× TBE running buffer to the upper and lower reservoirs.
• 50Rinse the wells thoroughly with 1× TBE to remove urea and nonpolymerized acrylamide.
• 51Prerun the gel at constant power (60–70 W) for approximately 30 min or until the gel reaches a temperature of 50–60 °C.
• 52Denature the samples/markers by heating to 95 °C for 5 min. As samples are heated, rinse the wells again with 1× TBE.
• 53Load samples and run gel at constant power (60–70 W) for 1.5–3 h or until the bromophenol blue dye (the faster moving dye) reaches the bottom of the gel.
• 54Disassemble the electrophoresis apparatus and carefully remove one of the glass plates so that the gel remains attached to the other. Transfer the gel from the glass plate by blotting onto a sheet of 3 MM Whatman filter paper. Cover the gel in plastic wrap and remove any excess gel and/or filter paper with scissors.
• 55Place the gel in a gel dryer sandwiched between two additional sheets of 3 MM Whatman filter paper and dry for 1 h under heat (~85 °C) and vacuum.

  Ensure that the gel has completely dried before releasing the vacuum seal. Incomplete drying may cause the gel to crack.

• 56Expose the gel using a PhosphorImager screen.

  Exposure time varies depending upon the specific activity of the DNA substrate. Normally exposure times range from 1 to 48 h.

• 57Scan the image using a PhosphorImager scanner.

Analysis/quantification of results

• 58In general, much of the analysis can be accomplished by inspecting the gel images and visually comparing the band intensities by eye. Alternatively, the percentage of inhibition (i.e., cleavage) may be calculated by two related methods using a gel imaging software, such as ImageQuant (Molecular Dynamics) (methods A and B).

  1. Measuring an increase in product signal

     1. In ImageQuant, manually draw two boxes around the full-length DNA band (box 1) and the DNA fragment bands (i.e., theentire lane excluding the full-length DNA band) (box 2).

     2. After subtracting the background, determine the total amount of radioactive signal in a given lane by summation of thesignals from box 1 and 2.

     3. Use the equation shown below to calculate the percentage of cleavage for a given lane.

        $$\begin{gathered} \mathrm{\%}\text{cleavage}=\left[\frac{\left(\text{signal}\text{for}\text{all}\text{DNA}\text{fragments}\right)}{\left(\text{total}\text{signal}\right)}\right]\times 100\mathrm{\%} \\ =\left[\frac{\mathrm{b}\mathrm{o}\mathrm{x}2}{(\text{box}1+\text{box}2)}\right]\times 100\mathrm{\%} \end{gathered}$$

        In this method, 50% inhibition is achieved when the sum of the radioactivity from the fragments is equal to that of the remaining full-length DNA.

  2. Measuring a decrease in substrate signal

     1. Use band intensities from box 1 (see Step 58A) and the equation below to calculate the percentage of cleavage.

        $$\begin{gathered} \mathrm{\%}\text{cleavage}=\left[\frac{(\text{signal}\text{for}\text{full}-\text{length}\text{DNA}\text{band}\text{of}\text{drug-treated}\text{lane})}{(\text{signal}\text{for}\text{full}-\text{length}\text{DNA}\text{band}\text{of}\text{no}\text{drug}\text{control}\text{lane})}\right]\times 100\mathrm{\%} \\ =\left[\frac{\text{box}{1}_{\text{drug-treated}}}{\text{box}{1}_{\text{no}\text{drug}\text{control}}}\right]\times 100\mathrm{\%} \end{gathered}$$

        In this method, 50% inhibition is achieved when the radioactivity from the full-length DNA band is reduced by half relative to the ‘no drug’ control full-length DNA band.

• 59
  We routinely express Top1 inhibition data semiquantitatively as follows (for examples, see refs. 77,80,81):

  Rank	Description
  0	No cleavage
  +	Between 20 and 50%, the cleavage activity of 1 μM CPT
  ++	Between 50 and 75%, the cleavage activity of 1 μM CPT
  +++	Between 75 and 100%, the cleavage activity of 1 μM CPT
  ++++	Equipotent to or more potent than 1 μM CPT

  IC50 values for a given inhibitor are better calculated using an oligonucleotide substrate containing a single high-affinity Top1 cleavage site.

  

  

  Troubleshooting advice can be found in Table 2.

TABLE 2 |.

Troubleshooting table.

Problem	Potential cause	Possible solution
Extensive banding in ‘no enzyme’ control lane	DNA substrate degraded	Repurify and relabel DNA substrate
Bands very faint in all lanes, including the band representing the full-length DNA substrate	Specific activity of DNA substrate is too low per reaction	Add at least 100k c.p.m. of radiolabeled DNA substrate to each reaction
	Radioisotope decay	Relabel with newly purchased radionucleotide
An absence of cleavage products; only full-length DNA substrate band is detectable, even in positive control	Top1 concentration too low	Increase the amount of Top1 per reaction
	Activity of enzyme has decreased or degraded	Reorder or repurify Top1 enzyme from appropriate source
Full-length DNA substrate band absent and only the low molecular weight cleavage bands are visible in drug-treated lanes	Top1 concentration too high	Reduce the amount of Top1 per reaction
All bands smeared or streaked	Acrylamide polymerization is not homogeneous	Ensure quality of gel solution and catalysts (APS and TEMED)
Biphasic cleavage pattern or suppression of cleavage at high concentrations of drug	Drug may be a DNA intercalator (see example in ref. 86)	Use alternate assay to identify DNA intercalators (i.e., DNA unwinding assay88)

ANTICIPATED RESULTS

Top1-mediated DNA cleavage assay

A representative denaturing polyacrylamide gel from the Top1-mediated DNA cleavage assay carried out in the presence of the reference Top1 inhibitor, camptothecin (see Fig. 3a), and the new noncamptothecin indenoisoquinoline, NSC 724998 (see ref. 6) (see Fig. 4), is shown in Figure 6. The induction of Top1–DNA complexes by both drugs was dose dependent, as evidenced by the increased intensity of the DNA cleavage bands and/or the decrease in the relative intensity of the full-length DNA substrate band. Note that some Top1 inhibitors may cause a biphasic effect in the formation of DNA cleavage products (i.e., reappearance of the full-length DNA substrate at high concentrations of inhibitor), which is a common characteristic of strong DNA intercalators⁸⁶ (see TROUBLESHOOTING). Figure 6 also illustrates that DNA cleavage sites can be numerically mapped from the 5′-end of the DNA substrate (arrows and numbers to the right in of the gel) by comparison with Maxam–Gilbert sequencing markers. Bear in mind that different Top1 inhibitors may exhibit different base sequence preferences, as evidenced by differential patterns of drug-induced DNA cleavage sites. For example, CPT and NSC 724998 induced DNA cleavage complexes at several similar sites with differences in their relative intensities (sites 70, 92, 97 and 119). However, some cleavage sites are unique to NSC 724998 (sites 44, 62 and 107–110) or CPT (site 37). Indeed, these differences in sequence selectivity could ultimately lead to different pharmacological outcomes.

Figure 6 |.

Top1-mediated DNA cleavage assay. Equal amounts of recombinant

Top1 were incubated with the 3′-end-labeled DNA substrate in the presence (CPT and NSC 724998) or absence of drug. The reaction products were resolved on a denaturing polyacrylamide gel. Lane 1, Maxam–Gilbert AG sequencing reaction; lane 2, negative enzyme control; lane 3, negative inhibitor control; lanes 4–7, 0.01, 0.1, 1 and 10 μM of CPT; lanes 8–11, 0.01, 0.1, 1 and 10 mM of NSC 724998. Arrows and numbers indicate the sites of DNA cleavage.

Top1 cleavage complex reversal assay

An example of the reversal assay of Top1-mediated DNA cleavage is shown in Figure 7. This type of assay is predominantly performed with the purpose of estimating the stability of the Top1–drug–DNA ternary complex^54,55. In Figure 7, reversal was accomplished by the addition of 0.35 M NaCl subsequent to the formation of stable drug-induced Top1cc. Note that the concentrations of 0.1 and 10 μM for CPT and NSC 724998 were chosen, respectively, to ensure an initial DNA cleavage of similar intensity at time zero (Fig. 7, lanes 4 and 11), which is necessary so as to not misinterpret the reversibility of Top1cc when comparing different inhibitors. Figure 7 illustrates that the reversal of Top1–DNA cleavage complexes at site 92, which is targeted by both drugs, is similar, although differences in the efficiency of trapping Top1 cleavage complexes are also observed at sites 44 and 119. Greater stability of Top1 cleavage complexes can contribute to the greater cytotoxicity of top1 inhibitors⁹⁶. Alternatively, this assay can also be carried out using a synthetic oligonucleotide duplex containing a single Top1 cleavage site that is unique to the inhibitor under investigation to facilitate a more quantitative measurement of the half-life/kinetics of the covalent complex (e.g., see ref. 11).

Figure 7 |.

Top1–DNA cleavage complex reversal assay. After 20 min of incubation with 0.1 or 10 μM of CPT or NSC 724998, respectively, 0.35 M NaCl was added to induce the reversal of the Top1 cleavage complexes. Aliquots were taken and reactions were terminated at various time points: 0, immediately before NaCl addition (lanes 4 and 11), 0.5 min (lanes 5 and 12), 1 min (lanes 6 and 13), 2 min (lanes 7 and 14), 5 min (lanes 8 and 15), 10 min (lanes 9 and 16) and 20 min (lanes 10 and 17).