Scintillation Proximity Assay for Human DNA Topoisomerase I Using Recombinant Biotinyl-Fusion Protein Produced in Baculovirus-Infected Insect Cells

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

240, 185–196 (1996)

0348

Scintillation Proximity Assay for Human DNA Topoisomerase I Using Recombinant Biotinyl-Fusion Protein Produced in Baculovirus-Infected Insect Cells Claude G. Lerner1 and Anne Y. Chiang Saiki Abbott Laboratories, Pharmaceutical Products Research Division, 100 Abbott Park Road, Abbott Park, Illinois 60064-3500

Received February 27, 1996

DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid highthroughput format. We describe here a highthroughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyltopoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated during in vivo expression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of 3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein–DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis. q 1996 Academic Press, Inc.

The earliest stages of drug discovery involve identification of an appropriate biological target, followed by development of an assay capable of rapidly screening large numbers of highly diverse chemical entities for novel inhibitors. One approach to target selection capitalizes on the emergence of new biological targets as 1 To whom correspondence should be addressed at Abbott Laboratories, D47N-AP9A, 100 Abbott Park Road, Abbott Park, IL 600643500. Fax: (847) 938-6603. E-mail:[email protected].

the wealth of biomedical information, especially from genome sequencing projects, increases. Such new targets represent a tantalizing lure to the discovery of novel therapeutic agents. Another approach is to select a proven biological target and search for new classes of inhibitors. One way to enhance the likelihood of finding new inhibitors of established targets is to employ novel methods to screen highly diverse sources of chemical species. We report here development of a novel assay to screen for inhibitors of human DNA topoisomerase I, an established anticancer target. Human DNA topoisomerase I is a monomeric 91-kDa protein that catalyzes the relaxation of positively or negatively supercoiled DNA molecules. The relaxation, or swivel, activity of topoisomerase I alleviates superhelical tension that is generated during replication and transcription. Upon binding its DNA substrate, topoisomerase I transiently breaks one strand, forming a nick in the duplex. The break allows the helix to unwind, and the nick is subsequently repaired by rejoining the ends of the broken strand. The breakage and rejoining steps are transesterification reactions in which the hydroxyl group of the active-site tyrosyl residue nucleophilically attacks the DNA phosphodiester backbone, forming a transient covalent phospho-tyrosyl-enzyme–DNA complex. The energy of the phosphodiester bond is conserved in the covalent phosphotyrosyl ester linkage. Following unwinding of the helix, the break is repaired by a second transesterification, or religation, reaction in which the previously liberated hydroxyl group on the free strand attacks the enzyme– DNA ester linkage to reform the DNA phosphodiester backbone (for reviews, see 1–6). The plant alkaloid camptothecin (CPT2) is a potent 2 Abbreviations used: CPT, camptothecin; SPA, scintillation proximity assay; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; PBS, phosphate-buffered saline; DMSO, dimethyl sulf-

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0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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and specific anticancer agent that interferes with the religation step of the reaction and effectively traps the protein covalently on the DNA (for reviews, see 1, 3, 7). Putatively, the cytotoxic effect of CPT results from collision of replication forks with CPT-stabilized covalent protein–DNA complexes. These collisions then lead to formation of lethal DNA strand breaks as the replication fork passes through the nicked site (8, 9). Semisynthetic derivatives of CPT show encouraging results as antitumor agents in the treatment of colorectal cancer, small-cell, and non-small-cell lung cancers, as well as cervical and ovarian cancers (10). Traditional methods for assaying the activity of topoisomerases range from high-resolution one- and twodimensional agarose gel electrophoresis (11), which resolve DNA substrate, product, and intermediate species, to filter-binding assays, which measure the formation of covalent enzyme–DNA complexes (for review, see 12). Although powerful as analytical techniques, most of the methods require lengthy separation steps to resolve unreacted substrates, reaction products, and intermediates. To screen for new classes of inhibitors of human topoisomerase I, we developed a simple assay that does not require a separation step to detect both noncovalent and covalent binding of DNA to the enzyme. The method employs the scintillation proximity assay (SPA) (13), which is readily amenable to highthroughput screening, including high-capacity robotic automation. The scintillation proximity assay utilizes microsphere beads that contain fluorescent scintillant embedded within the beads. In aqueous solution the lowenergy beta particles emitted by 3H-atoms rapidly lose their energy through collisions with water molecules. The maximal distance traveled by a tritium decay beta particle in aqueous solution is about 4 mm. When a tritiated solute is placed in aqueous solution with SPA microsphere beads, very few beta particles of sufficient energy are able to excite the scintillant. However, if the tritiated solute is retained in close proximity to the bead (Ç1.5 mm), many beta particles of sufficient energy collide with and excite the scintillant, resulting in significant light emission from the beads. The topoisomerase I scintillation proximity assay described here allows detection of enzyme–DNA binding by using biotinylated topoisomerase I immobilized on streptavidin-coated SPA beads. Binding of 3H-labeled plasmid DNA to the bead-immobilized enzyme is detected by scintillation counting as an increase in the amount of radioactivity observed. Inhibitors that preoxide; AcNPV, Autographa californica nuclear polyhedrosis virus; BCCP, biotin carboxyl carrier protein; AcNPV/BCCP-TOP1, recombinant AcNPV virus containing biotinyl-human topoisomerase I gene fusion; PMT, photomultiplier tube; DTT, dithiothreitol.

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vent DNA binding result in a decrease in the amount of radioactivity detected. Such inhibitors would prevent enzyme catalysis. Covalent complex stabilizers, such as CPT, are detected by examining the amount of [3H]DNA bound after addition of SDS, which disrupts noncovalent enzyme–DNA interactions. Such inhibitors give rise to an increase in the signal after SDS addition. A third type of inhibitor, which interferes with covalent complex formation but does not affect noncovalent enzyme–DNA interactions, can also be detected by the assay. Such inhibitors would allow [3H]DNA binding to be detected only in the absence of SDS. This class of inhibitors would be predicted to alter enzyme catalysis but not to interfere with substrate binding. Biotinylated topoisomerase I protein was prepared by fusing the human TOP1 gene encoding topoisomerase I at its amino terminus to a gene encoding the biotin-accepting domain of a bacterial biotin carboxyl carrier protein (BCCP). Fusion of the BCCP domain to topoisomerase I allows endogenous enzymes to biotinylate the fusion protein in vivo. The biotin group is attached through an amide bond to an e-amino group of a lysine residue in the BCCP domain (14–16). The covalently attached biotin provides a means of fixing the enzyme to streptavidin-coated SPA beads. We have expressed recombinant biotinyl-fusion protein in baculovirus-infected insect cells to develop a rapid SPA-based assay to detect DNA binding to human DNA topoisomerase I. Our studies show that this assay can be used to assess rates of DNA association and dissociation. It can also be used to detect compounds that stabilize covalent DNA–enzyme intermediates that form during the enzymatic reaction. MATERIALS AND METHODS

Reagents. Camptothecin was purchased from Sigma (St. Louis, MO). Tissue culture media for the growth of Spodoptera frugiperda (Sf9) insect cells were purchased from PharMingen (San Diego, CA). Supercoiled PM2 DNA was purchased from BoehringerMannheim (Indianapolis, IN). All other chemicals were of reagent grade. Streptavidin-coated SPA microsphere beads were purchased from Amersham (Arlington Heights, IL). Cells and plasmids. The baculovirus transfer vector pVL1392, BaculoGold DNA, baculovirus strain Autographa californica nuclear polyhedrosis virus (AcNPV), and S. frugiperda (Sf9) insect cells were purchased from PharMingen. The plasmid YEpGAL1hTOP1 containing the human TOP1 gene encoding DNA topoisomerase I was obtained from Dr. James C. Wang, Harvard University (17). Plasmid pBluescript KS/ (pBSKS/) was purchased from Stratagene (La Jolla, CA). AL3H5a is a thymine-requiring mutant de-

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rived from Escherichia coli strain DH5a by selection for resistance to trimethoprim (18). DH5a was obtained from Gibco BRL (Gaithersburg, MD). Plasmid PinPoint Xa3 (pPPXa3), which contains the gene encoding the 1.3S biotin-containing transcarboxylase BCCP subunit from Propionibacterium shermanii, was purchased from Promega (Madison, WI). Plasmid pET-23(/) was purchased from Novagen (Madison, WI). Human biotinyl-topoisomerase I fusion protein preparation. Expression of biotinylated human topoisomerase I fusion protein in Sf9 insect cells was accomplished utilizing the BaculoGold baculovirus expression system from PharMingen. The procedures detailed in the manual provided by the supplier were followed. Recombinant AcNPV virus (AcNPV/BCCPTOP1), which contains the human TOP1 gene fused to the biotinylation domain (BCCP-TOP1) and expressed from the strong polyhedrin promoter of AcNPV, was generated using the pVL1392-BiohTOP1 transfer vector described below. The recombinant AcNPV/BCCPTOP1 virus was used to infect Sf9 cells for production of the desired fusion protein. Experimental controls included uninfected Sf9 cells and wild-type AcNPV-infected cells. The transfer vector plasmid pVL1392-BiohTOP1 was constructed for production of biotinyl-topoisomerase I fusion protein. A Ç2.5-kb EcoRV–BamHI fragment containing the TOP1 gene was isolated from plasmid YEpGAL1-hTOP1 and cloned into the EcoRV and BamHI sites of pPPXa3. The resulting plasmid was designated pPPXa3-hTOP1 and contains an in-frame fusion of the gene fragment encoding the BCCP domain to the start codon of the TOP1 gene. A second intermediate was constructed by cloning a Ç3.0-kb EcoRI– NotI fragment containing the BCCP-TOP1 gene fusion from pPPXa3-hTOP1 into the EcoRI and NotI sites of pET-23(/). The resulting plasmid was designated pET23-BiohTOP1. The reading frame of the gene fusion was confirmed in both constructs by DNA sequencing. The final plasmid, pVL1392-BiohTOP1, was constructed by cloning a Ç2.9-kb PstI–BglII fragment containing the BCCP-TOP1 gene fusion from pET23BiohTOP1 into the PstI and BamHI sites of the plasmid pVL1392. The BCCP domain adds 138 amino acid residues to the native enzyme. The resulting fusion protein is predicted to be 903 residues in length and have a molecular mass of 103,787 daltons. Approximately 72 h postinfection, Sf9 cells were harvested and washed with PBS. To determine the localization of the biotinyl-topoisomerase I fusion protein, the cells (including wild-type AcNPV and uninfected controls) were fractionated according to a protocol adapted from Miyamoto et al. (19). The cells were incubated in hypotonic buffer (10 mM Tris – HCl,

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pH 7.5; 10 mM NaCl; and 1.5 mM MgCl2) at 47C for 30 min and were centrifuged to separate the nuclei from the cytoplasmic fraction. After removal of the supernatant (cytoplasmic fraction), the nuclear pellet was incubated in hypotonic buffer containing 1% Nonidet P-40 and 0.5% deoxycholate at 47C for 30 min to strip the nuclei. After centrifugation, the supernatant containing the membrane-associated fraction was removed. To dissociate the biotinyl-topoisomerase I fusion protein from the chromatin and to swell the nuclear pores, the nuclei were then incubated in hypotonic buffer containing 0.5 M NaCl at 47C for 30 min. After centrifugation, the supernatant (nuclear fraction) containing the biotinylated human topoisomerase I fusion protein was removed, and the insoluble pellet was resuspended in hypotonic buffer. The nuclear fractions were diluted in 21 storage buffer (99% glycerol; 100 mM NaCl; 100 mM K2HPO4KH2PO4 , pH 7.0; 0.1 mM PMSF; and 1 mM DTT), aliquoted, and stored at 0207C. At appropriate points during the fractionation, samples of the fractions were taken and analyzed by SDS – PAGE (20). The presence of biotinyl-topoisomerase I fusion protein in the nuclear fraction was confirmed by Western blot analysis (21) with an avidin – alkaline phosphatase conjugate probe. Protein concentrations were determined with BSA as a standard by the method of Bradford (22). Preparation of [3H]pBSKS/ DNA. Tritium-labeled plasmid DNA was prepared by culturing a transformant of AL3H5a containing the plasmid pBSKS/ in Terrific Broth medium (20) with 270 mCi/ml [3H]methylthymidine (Ç80 Ci/mmol), 9 mg/ml cold thymidine, and 100 mg/ml carbenicillin at 377C for 16–19 h with vigorous aeration. 3H-labeled plasmid DNA was purified from the cells using a plasmid isolation kit (QIAGEN, Chatsworth, CA) in accordance with the supplier’s instructions. The resulting [3H]pBSKS/ plasmid DNA had a specific activity of 2–5 mCi/mg and was determined to be in Ç95% supercoiled form by agarose gel electrophoresis. Topoisomerase I scintillation proximity assays. Biotinyl-topoisomerase I fusion protein was immobilized onto the surface of streptavidin-coated SPA beads by diluting the enzyme preparation in binding buffer (100 mM NaCl; 20 mM Tris–HCl, pH 7.5; 1 mM EDTA; 0.5 mM DTT; 30 mg/ml BSA). Concentrated streptavidincoated SPA beads (20 mg/ml in PBS; Ç100 pmol biotin/ mg binding capacity) were then added to a final concentration of 1.25–2.5 mg/ml. The tube was capped and incubated at 47C for 60 min with gentle mixing on a rocking platform. Unbiotinylated impurities were separated from the enzyme-coated SPA beads by filtration through a 0.45-mm low-protein-binding filter (Millipore

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Ultrafree-CL Durapore, Millipore, Bedford, MA). The suspension was transferred to a filter unit which was prerinsed with binding buffer to block residual protein binding sites. Unbound material was removed by brief centrifugation in a swinging bucket rotor by rapid acceleration up to Ç1000g (Ç30 s). The beads were resuspended in 2 ml ice-cold high-salt wash buffer (1 M NaCl; 20 mM Tris–HCl, pH 7.5; 1 mM EDTA; 0.5 mM DTT; 30 mg/ml BSA). The high-salt wash was repeated a total of three times. The beads were then rinsed once with 2 ml reaction buffer (5 mM MgCl2 ; 20 mM Tris–HCl, pH 7.5; 1 mM EDTA; 0.5 mM DTT; 30 mg/ml BSA) and resuspended in 2 ml of the same buffer. Topoisomerase–SPA reactions were performed by mixing 40–80 ml of biotinyl-topoisomerase I-coated SPA beads (Ç100 mg beads, 20 U enzyme activity) with [3H]pBSKS/ in a total of 100 ml. The reactions were prepared in microcentrifuge tubes or 96-well microplates (OptiPlate, Packard, Meriden, CT) and incubated at room temperature (237C) for the times indicated. The microplate was sealed with plastic film, and the amount of radioactivity in the wells was determined using a Packard TopCount microplate scintillation counter in high efficiency dpm 3H-liquid scintillator mode. Samples were counted for 3.5 min or until the counting error was £3.6% (2 sigma % count statistic). Color quenching was corrected using a quench curve prepared with tartrazine dye according to the manufacturer’s instructions. The resulting quench-corrected cpm were designated qc-cpm. Continuous topoisomerase–SPA reactions were performed in microcentrifuge tubes. [3H]DNA binding was assessed by placing the tube in a conventional scintillation counter (Packard TriCarb 1900 TR) using a microtube adapter. Readings were taken over 0.1-min intervals. The amount of [3H]DNA covalently bound to the enzyme was assessed after quenching the reaction with 50 ml of 1.5% SDS (0.5% final). The efficiency of counting 3H-SPA samples on the TopCount microplate counter, which utilizes a single PMT per well, was 43% relative to counting the same samples on the TriCarb, which uses two-PMT coincidence counting. CPT stocks were freshly prepared with 100% DMSO. Experiments with CPT contained a final concentration of 1% DMSO in the reaction. The presence of 1% DMSO in the reaction had no effect on the SPA signal (data not shown). Topoisomerase relaxation assay. Conversion of supercoiled PM2 DNA to its relaxed form was measured as follows. Serial twofold dilutions of the protein fractions were prepared in buffer consisting of 10% glycerol, 15 mM KPO4 (pH 7.2), 0.1 mM EDTA, and 1 mg/ ml BSA and were assayed in reaction buffer consisting of 100 mM KCl, 10 mM MgCl2 , 20 mM Tris (pH 7.5), 0.1

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mM EDTA, and 40 mg/ml BSA. After preequilibration at 377C for 3 min, the diluted protein samples (5 ml) were transferred to reaction buffer (15 ml) containing 125 ng supercoiled PM2 DNA. After incubation at 377C for 15 min, the reactions were stopped by addition of 5 ml gel loading buffer containing 5% SDS. The amount of conversion of supercoiled substrate to relaxed product was determined by agarose gel electrophoresis. Following electrophoresis, the gels were stained with 0.5 mg/ ml ethidium bromide, and the DNA was visualized by uv transillumination. A negative image of the gel was scanned using a Molecular Dynamics personal densitometer Model SI (Sunnyvale, CA). The apparent amount of DNA in individual bands was quantitated using ImageQuaNT image analysis software from Molecular Dynamics. The unit definition is the activity which will convert one-half of the 125 ng of supercoiled PM2 DNA substrate into the relaxed form at 377C in 15 min. RESULTS

Production of Biotinylated Human Topoisomerase I Fusion Protein in Recombinant BaculovirusInfected Insect Cells The TOP1 gene encoding human DNA topoisomerase I was fused to the carboxyl-terminal portion of a BCCP gene in two bacterial expression vector systems utilizing the tac or T7 promoters. Expression of the hybrid fusion protein was not detected in E. coli (data not shown). While it has been difficult to produce significant quantities of native human topoisomerase I in E. coli (17, 23), the use of recombinant baculovirus expression vectors in insect cells has facilitated production of the protein (24–26). A recombinant AcNPV/BCCPTOP1 baculovirus stock containing the BCCP-TOP1 gene fusion expressed from the strong polyhedrin promoter was constructed and used to infect Sf9 insect cells. Three days after infection, the cells were harvested and fractionated into nuclear, cytoplasmic, membrane-associated, and insoluble fractions. Fractions from wild-type AcNPV-infected and uninfected cells were also prepared and analyzed as controls. Figure 1A shows a Coomassie blue-stained SDS–PAGE analysis of the fractions. One new band corresponding to a protein with a molecular mass of Ç100 kDa appears in the nuclear fraction from the recombinant AcNPV/BCCP-TOP1 infected cells. A Western blot of a duplicate SDS–polyacrylamide gel was probed with a streptavidin–alkaline phosphatase conjugate, and a unique biotinylated species with a molecular mass of Ç100 kDa was detected exclusively in the nuclear and cytoplasmic fractions of the recombinant virus-infected cells (Fig. 1B). This is consistent with the expected molecular mass (104 kDa) of the biotinyl-toposiomerase I

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immobilized enzyme was found to be 3 1 104 units of DNA relaxing activity per milligram of total nuclear extract protein originally present in the capture incubation with the streptavidin-coated beads. A total of 0.4 mg nuclear extract protein (Ç1.2 1 104 units immobilizable activity) was obtained from 4 1 107 Sf9 cells infected with a multiplicity of infection of Ç1. Scintillation Proximity Assay with Human DNA Topoisomerase I Biotinyl-topoisomerase I-coated SPA beads were prepared and assayed for DNA binding activity with [3H]pBSKS/ plasmid DNA (Fig. 2). The SPA signal was linear up to at least 40 units of bead-immobilized enzyme from 1.5 mg of input nuclear extract. The background SPA signal observed with uncoated streptavidin-SPA beads as a control was õ90 qc-cpm. The observed signal-to-noise ratio was Ç200-fold above background. The signal decreased after addition of 0.5% SDS, which dissociated noncovalent enzyme–DNA complexes. The residual SPA signal is from latent covalent enzyme–DNA complexes. The error between duplicate samples was õ10%. Control reactions were performed with beads prepared with an amount of nuclear extract from wild-type AcNPV-infected cells equivalent to the amount of recombinant AcNPV/BCCP-

FIG. 1. Analysis of expression and localization of in vivo biotinylated human DNA topoisomerase I fusion protein in baculovirus-infected Sf9 insect cells. Wild-type AcNPV- and recombinant AcNPV/ BCCP-TOP1-infected and uninfected Sf9 cells were fractionated as described in the text. (A) Coomassie blue-stained SDS–polyacrylamide gel. (B) Western blot probed with an avidin–alkaline phosphatase conjugate.

fusion protein. When normalized to total cell number, enzyme assays with the nuclear fractions indicated 10fold more DNA relaxing activity in the nuclear extract from the recombinant AcNPV/BCCP-TOP1 virus-infected cells than extracts from the wild-type AcNPVinfected or uninfected cells (data not shown). These results indicate the biotinyl-topoisomerase I fusion protein is overproduced, active, and correctly localized to the nuclear fraction. It is also the most prominent biotinylated protein in the nuclear fraction. The catalytic activity of the biotinyl-topoisomerase I fusion protein immobilized on streptavidin-coated SPA beads was also analyzed. The apparent activity of the

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FIG. 2. Dependence of topoisomerase–SPA signal on concentration of biotinyl-topoisomerase I-coated SPA beads. Binding reactions contained 3H-plasmid DNA (0.25 mCi, 0.5 mg/ml, 260 pM) and varying amounts of enzyme-coated SPA beads in a total of 100 ml in a 96well microplate. After incubation for 20 min, the plate was sealed and radioactivity read in a microplate scintillation counter. SPA signals detected from total (noncovalent and covalent) [3H]DNA–enzyme complexes (h) and from covalent complexes stable to denaturation with 0.5% SDS (s) are shown. Data points shown are the average of duplicate samples.

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Binding of 3H-Plasmid DNA to Immobilized Biotinylated Topoisomerase I Assessed by Scintillation Proximity Assay Binding of 3H-plasmid DNA to the biotinyl-topoisomerase I fusion protein was examined by continuous

FIG. 3. Dependence of topoisomerase–SPA signal on 3H-plasmid DNA substrate concentration. Reactions containing 200 units/ml of biotinyl-topoisomerase I-coated SPA beads with various amounts of 3 H-plasmid DNA were incubated for Ç2 h in reaction buffer containing 150 mM NaCl. The plate was sealed and radioactivity read in a microplate scintillation counter. SPA signals from noncovalent and covalent enzyme–[3H]DNA complexes (h) and from covalent complexes resistant to denaturation with 0.5% SDS (n) are shown. SPA signals from control reactions performed with beads prepared with an amount of nuclear extract from wild-type AcNPV-infected cells equivalent to the amount of recombinant AcNPV/BCCP-TOP1 extract that gave 200 units/ml activity in the absence (L) and presence (1) of 0.5% SDS are also shown. Data points shown are the average of duplicate samples with error bars indicating the standard deviation.

TOP1 extract that gave 20 U of enzyme activity. The resulting signals with these control beads were 700 and 0 qc-cpm in the absence and presence of SDS, respectively. The dependence of the topoisomerase-SPA signal on [3H]DNA substrate concentration was also examined. Figure 3 shows a plot of [3H]DNA–enzyme complex formation as a function of total [3H]DNA concentration. At higher [3H]DNA concentrations the background signal observed with uncoated control beads became similar to the signal observed with enzyme-coated beads indicating loss of the scintillation proximity effect. The stability of bead-immobilized enzyme was examined by assessing the [3H]DNA binding activity of beads stored as a twofold concentrated suspension at 47C. After 24 h of storage, the beads retained 90% of their original activity. Prolonged storage indicated a logarithmic loss of activity with 50% of the activity remaining after 10 days of storage (data not shown). For the experiments shown here, enzyme-coated beads were freshly prepared from nuclear extracts stored at 0207C in 50% glycerol.

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FIG. 4. Kinetics of 3H-plasmid DNA binding to biotinyl-topoisomerase I-coated SPA beads. (A) [3H]DNA (260 pM, 0.5 mg/ml, 0.25 mCi) was quickly added to a microcentrifuge tube containing enzymecoated SPA beads in reaction buffer with 150 mM NaCl. The tube was rapidly placed in a scintillation counter set to continuously monitor radioactivity over 0.1-min time intervals. The extent of binding of [3H]DNA to 200 (n), 100 (h), and 50 (s) units/ml of enzyme-coated SPA beads is shown. Control reactions containing uncoated SPA beads for assessment of nonspecific binding are also shown (L). (B) Time course of covalent enzyme–[3H]DNA complex formation. Reactions were quenched by addition of 0.5% SDS at the times indicated. The extent of covalent binding of 3H-plasmid DNA (260 pM, 0.5 mg/ml, 0.25 mCi) to 200 units/ml of enzyme-coated SPA beads as a function of time was monitored by scintillation counting (s).

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FIG. 5. Kinetics of enzyme–[3H]DNA complex dissociation. Excess unlabeled plasmid DNA (70 mg/ml; 36 nM) was added to equilibrated reactions set up as in Fig. 3 with 260 pM [3H]DNA (0.5 mg/ml, 0.25 mCi) and 200 units/ml enzyme-coated beads in reaction buffer with 150 mM NaCl. The dissociation of [3H]DNA was monitored continuously over 0.1-min intervals by scintillation counting (s).

analysis of [3H]DNA interaction with enzyme-coated SPA beads. Figure 4A shows binding of [3H]DNA to various amounts of enzyme-coated beads. No increase in signal was observed for control reactions containing uncoated beads. The observed rate of [3H]DNA binding reflects formation of both covalent and noncovalent enzyme–DNA complexes. To examine covalent complex formation exclusively, reactions were quickly quenched by addition of 0.5% SDS at specific times after [3H]DNA addition. Figure 4B shows a plot of the time course of covalent enzyme–DNA complex formation. To examine the dissociation of topoisomerase–DNA complexes, topoisomerase–SPA reactions were incubated for 2 h, at which point equilibrium steady-state binding was achieved. A large excess of unlabeled pBSKS/ plasmid DNA was then added to the mixture. Figure 5 shows a plot of the dissociation of [3H]DNA– enzyme complexes over time. As a control, an equal volume of buffer was added to a second aliquot of beads. No decrease in signal was observed (data not shown). The decrease in signal was thus due to dissociation of the [3H]DNA–enzyme complexes by competition with unlabeled plasmid DNA rather than dilution of the sample volume. Detection of Camptothecin Enhancement of Topoisomerase I Covalent Complex Stabilization by Scintillation Proximity Assay The effect of the potent covalent complex stabilizer, camptothecin, on the topoisomerase–SPA reaction was

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also examined. Reactions containing various amounts of CPT were prepared. The radioactivity from both covalent and noncovalent complexes in the reactions was assessed in a microplate scintillation counter (Fig. 6A). To correct for sample-to-sample variation in the amount of [3H]DNA or enzyme-coated beads in the reaction, the fraction of counts remaining after SDS addition was calculated (covalent complex fraction) and plotted as a function of CPT concentration (Fig. 6B). Camptothecin-induced covalent complex stabilization is apparent from the [3H]DNA binding data (Fig. 6A). Correspondingly, the covalent complex fraction increases from 0.33 to approximately 0.53 with increasing CPT concentration. The CPT concentration which gives rise to one-half of the maximal observed covalent complex stabilization ratio was 1.5 mM under the conditions tested. The effect of ionic strength on covalent complex formation was examined by performing topoisomerase– SPA reactions in the presence of increasing concentrations of NaCl (Fig. 7). Total complex (noncovalent and covalent) formation was independent of salt concentration until 100 mM NaCl, after which a decrease in the signal is observed. At 300 mM NaCl the signal decreased by 50%. An essentially identical pattern was observed for similar reactions containing 50 mM CPT. Covalent complex formation, reflected in readings taken after SDS addition, increases by Ç33% with increasing NaCl concentration until 100 mM, after which a decrease in the signal is observed. NaCl concentrations up to 100 mM only slightly stimulated formation of CPT-stabilized covalent complexes. CPT stabilization was apparent at NaCl concentrations from 100 to 200 mM, but at 300 mM its effect was antagonized. The effect of salt addition to reactions containing preformed CPT-stabilized complexes was also examined by the topoisomerase-scintillation proximity assay (Fig. 8). High concentrations of NaCl have been found to reverse CPT-induced covalent complexes (27). Reactions containing [3H]DNA and enzyme-coated SPA beads { 50 mM CPT without added NaCl were prepared. The extent of formation of both covalent and noncovalent complexes, as well as only covalent complexes, was assessed after addition of 0.5 M NaCl or an equal volume of H2O. CPT-stabilized covalent complexes were reversed by addition of high salt, but were resistant to addition of H2O. Addition of 0.5 M NaCl decreases the amount of [3H]DNA bound in both covalent and noncovalent complexes to comparable levels in the presence or absence of 50 mM CPT. DISCUSSION

We have developed a novel method to detect formation of DNA–topoisomerase I complexes using the scin-

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FIG. 6. Effect of camptothecin on binding of 3H-plasmid DNA to biotinyl-topoisomerase I-coated SPA beads. Reactions containing 260 pM 3 H-plasmid DNA (0.5 mg/ml, 0.25 mCi) and 200 units/ml enzyme-coated beads were incubated for 20 min with various concentrations of CPT in a 96-well microplate. The plate was sealed and radioactivity read in a microplate scintillation counter. (A) SPA signals from noncovalent and covalent enzyme–DNA complexes (h) and from SDS-resistant covalent complexes (n). (B) Data from A represented as a covalent complex fraction, the fraction of total qc-cpm that are stable to SDS denaturation (s). Data points shown are the average of duplicate samples with error bars indicating the standard deviation.

tillation proximity assay. Human topoisomerase I was expressed from a baculovirus vector as a biotinyl-fusion protein. As expected, the fusion protein was predominantly nuclear-localized since the native enzyme contains a nuclear localization motif (28). Production of hybrid-biotinyl fusion protein using a baculovirus expression system has been previously reported (29). However, biotinyl-topoisomerase I appears to be the first instance of a nuclear-localized biotinyl-fusion protein. Cronan originally proposed using fusion proteins containing bacterial BCCP domains to study protein trafficking and localization in eukaryotic cells (14). Bacterial BCCP domains have been found to be biotinylated in both Saccharomyces cerevisiae (14, 30) and S. frugiperda (Sf9) insect cells (29). In particular, addition of a bacterial BCCP domain did not interfere with proper localization of the ATP11 protein found in mitochondria of S. cerevisiae (30). While there are only limited examples of production of hybrid fusion proteins containing bacterial BCCP domains in higher eukaryotic cells, it seems likely that such an approach could be generally used in determining the localization of other proteins. Given the wide distribution and crossspecies activity of biotin ligase in cells that perform carboxylation and decarboxylation reactions (31, 16), use of bacterial BCCP domains should be suitable for such studies. Use of a eukaryotic BCCP biotin-accepting domain such as the human propionyl-CoA carboxylase a subunit (16) may be preferable. Since biotinyl-topoisomerase I appeared to be the only biotinylated protein in the nuclear fraction, fur-

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ther purification of the enzyme was not necessary. The biotinyl-topoisomerase I fusion protein could be captured from crude nuclear extracts using streptavidincoated SPA beads. Contaminating nuclear proteins were removed by washing the enzyme-coated beads after collecting them on low protein-binding 0.4-mm filters. The bead-immobilized enzyme from the nuclear extract was catalytically active. The apparent specific activity of the enzyme, partially purified to greater than 90% apparent homogeneity by monomeric avidin affinity chromatography (A. Saiki and C. Lerner, unpublished results; 32), was approximately 1–2 1 106 units/mg, a value similar to those previously reported for the native enzyme isolated from various sources (25, 26, 33–35). The topoisomerase-scintillation proximity assay signal originates from both covalent and noncovalent binding of [3H]DNA to the SPA bead-immobilized enzyme. During the course of the analysis the supercoiled [3H]DNA substrate is enzymatically converted to relaxed product. Thus, the noncovalent [3H]DNA–enzyme complexes consist of both supercoiled and relaxed [3H]DNA with the majority of the enzyme bound to relaxed species shortly after initiation of the reaction. This makes the binding studies difficult to interpret since the enzyme has been found to exhibit a strong preference for binding supercoiled DNA compared to relaxed circular DNA (26, 36–38). It has been proposed that topoisomerase I binds at nodes where two DNA duplexes cross and preferentially binds supercoiled DNA because it possesses more nodes than relaxed DNA (26).

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FIG. 7. Effect of NaCl concentration on binding of 3H-plasmid DNA to biotinyl-topoisomerase I-coated SPA beads in the presence and absence camptothecin. Reactions containing 260 pM 3H-plasmid DNA (0.5 mg/ml, 0.25 mCi) and 200 units/ml enzyme-coated beads were incubated with various concentrations of NaCl { CPT for 20 min in a 96-well microplate. The plate was sealed and radioactivity read in a microplate scintillation counter. SPA signals from noncovalent and covalent [3H]DNA binding complexes (h, j) and from SDS-resistant covalent complexes (n, m) in the presence (solid symbols) and absence (open symbols) of 50 mM CPT. Data points shown are the average of duplicate samples with error bars indicating the standard deviation.

About 30–60% of the total [3H]DNA bound to the bead-immobilized enzyme remains associated with the bead in the presence of 0.5% SDS. We assume this residual signal arises from the trapping of covalent [3H]DNA–enzyme complexes. Madden et al. analyzed binding of relaxed and supercoiled [3H]DNA by a filter binding method with both native enzyme and catalytically inactive protein containing a mutation of the active-site tyrosine to phenylalanine (26). Analysis of their data indicates that the mutant enzyme bound approximately 33% less [3H]DNA than the native enzyme. This suggests the possibility that one-third of the [3H]DNA bound to the native enzyme was covalently attached, a value similar to the fraction we detect in the topoisomerase-scintillation proximity assay. In the results shown here, the maximum amount of [3H]DNA bound to the bead-immobilized enzyme was only 5–15% of the total amount of [3H]DNA present in the reaction. An estimate of the approximate amount of enzyme that was immobilized on the beads was made from the apparent specific activity of the partially purified enzyme. Using this value, only 5–10% of the beadimmobilized enzyme appeared to have [3H]DNA bound at the highest testable DNA concentration. Given the

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number of units of enzyme activity in the reaction, the majority of the noncovalently bound [3H]DNA was likely of relaxed form soon after substrate addition. Continuous [3H]DNA binding experiments were performed to assess the kinetics of [3H]DNA association and dissociation. The data were mathematically modeled to curves defined by expressions containing single exponential terms. Analysis of the rate of covalent complex formation showed biphasic kinetics described by an equation containing both an exponential and a linear term. This suggests that there are two different populations of enzyme bound to the beads, one with rapid and the other with slow binding kinetics. This could be explained by differences in phosphorylation of the enzyme. Topoisomerase I has been found to be phosphorylated in vivo (39). The catalytic activity of the enzyme in vitro is enhanced by phosphorylation on serine and threonine residues, including the same amino acid residue identified from in vivo studies (40). It is possible that the slow kinetic phase detected in the scintillation proximity assay arises from covalent complexes formed with the less active, unphosphorylated form of the enzyme. An alternative explanation is that a population of enzymes are immobilized on the surface of the bead such that access of the substrate to the enzyme is hindered. This possibility indicates that

FIG. 8. Salt reversal of camptothecin-stabilized [3H]DNA–topoisomerase I complexes. Reactions containing 260 pM 3H-plasmid DNA (0.5 mg/ml, 0.25 mCi) and 200 units/ml enzyme-coated SPA beads {50 mM CPT were prepared and incubated for 20 min. Onetenth volume of 5 M NaCl or H2O was added to the reactions. The extent of complex formation was assessed after addition of 0.5 M NaCl or an equal volume of H2O before (open bars) and after (shaded bars) dissociation of noncovalent complexes by addition of 0.5% SDS. Data values shown are the average of duplicate samples with error bars indicating the standard deviation.

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the method may have limited sensitivity compared to assays employing enzymes free in solution. The method was also able to detect CPT-stabilization of covalent enzyme–DNA complexes. When CPT concentrations were increased, an increase in the fraction of [3H]DNA bound in the presence of SDS was observed, although total DNA binding was not significantly effected. These results are consistent with those previously reported in which a filter-binding assay was used to detect DNA–enzyme complexes (41). In filterbinding reactions containing 2 nM enzyme, an amount similar to that present in our assays, addition of 5 mM CPT increased the fraction of SDS-stable covalent complexes by Ç20% (41). Consistent with their results, we found that CPT increases the stabilization ratio in the SPA–topoisomerase I reactions by 20% at concentrations §5 mM. The limited increase in CPT-induced covalent complex stabilization detected by the topoisomerase–SPA method suggests that it would be difficult to identify agents less potent than CPT using the reaction conditions as described here. It is possible that increasing the amount of enzyme used in the assay would lead to stronger signals and a more sensitive assay. Camptothecin-induced covalent complexes have been shown to be reversible. In particular, high salt concentrations dramatically reduce CPT-induced single-strand breaks that result from stabilized covalent complexes (27). The rate of salt-mediated reversal has been found to be dependent on both the DNA sequence of the cleavage site and the structure of the CPT analog. Furthermore, it has been suggested that resistance to salt reversal may be relevant to the stability of druginduced covalent complexes under physiological conditions (8). We were able to observe salt-mediated reversal of CPT-stabilized covalent complexes in SPA–topoisomerase I reactions. It has been suggested that development of CPT derivatives with extremely slow reversal kinetics may lead to highly active anticancer agents (8). In particular, a positive correlation has been observed between the cytotoxicity of CPT derivatives and several biochemical characteristics, including (i) the potency of induction of enzyme-mediated DNA cleavage, (ii) inhibition of enzyme activity, (iii) cellular DNA damage, and (iv) the kinetics of the reversal of drug-induced single-strand breaks in isolated nuclei (42). The topoisomerase I scintillation proximity assay may be a useful technique for the analysis of salt reversal kinetics of such analogs. Advantages and Potential Applications of BiotinylFusion Protein Technology for Development of Novel Scintillation Proximity Assays Several problems associated with in vitro biotinylation reactions using conventional chemical modification

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reagents were avoided by use of biotinyl-fusion protein technology. Disadvantages of chemical modification can include (i) modification of amino acid residues important for protein function, resulting in alteration of target protein activity; (ii) difficulty in controlling the extent of reaction, which must be monitored carefully; or (iii) requirement of relatively large amounts of pure protein. In particular, modification of large proteins frequently produces heterogeneous mixtures. Hyper- or hypomodification could adversely affect the reproducibility and sensitivity of an assay. These problems are readily alleviated by in vivo production of biotinyl-fusion proteins that contain a biotin moiety covalently attached at a single, specific lysine residue. It is likely that use of biotinyl-fusion protein technology would allow additional target proteins to be rapidly incorporated into SPA-formatted screens. Assays for proteins that lack catalytic activity but interact with therapeutically important ligand molecules, such as hormones, peptides, proteins, or nucleic acids, would be well-suited to this method. Examples of such proteins include DNA binding proteins such as transcription factors, RNA binding proteins, soluble domains of receptors, and proteins that bind to other proteins to form stable complexes such as cell adhesion molecules. Proteins that have been shown to be functional when fused to the 223-amino-acid glutathione S-transferase (GST) domain could also be readily incorporated into the assay. Hybrid fusion proteins in which the GST domain was replaced by the smaller 75- to 138-amino-acid biotinyl-fusion domain would likely retain activity. As shown here for camptothecin-induced stabilization of covalent topoisomerase I–DNA complexes, the method may be used to identify an interesting class of inhibitors of enzymes which form covalent intermediates during catalysis. By screening for compounds that show an increase in the SPA signal after quenching with a denaturant, compounds that stabilize the covalent intermediate may be identified. Screens that yield a positive signal in the presence of an inhibitor are highly desirable since they are likely to identify specific agents. Nonspecific protein denaturants that cause a decrease in signal by general inactivation of the target protein are frequently detected by negative signal screens. A positive signal-based assay would save time and resources, especially during natural product isolations. While the absence of sequence information may limit the application of this method, it may not be necessary in the case of membrane-associated target proteins, such as receptors or drug efflux pumps. It is likely that the method would allow such target proteins to be configured into the scintillation proximity assay via linkage of biotinylated whole cells or membrane preparations to streptavidin-coated SPA beads. Biotinylated

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whole cells can likely be prepared by expressing a hybrid fusion protein consisting of the BCCP domain covalently connected to a membrane localization protein sequence such that it becomes anchored to the membrane leading to stable attachment of the biotinyl domain to one or the other side of the cytoplasmic membrane. Proteins that require a free amino-terminal residue for activity present another challange. However, such proteins may be incorporated into the method via construction of biotinyl-domain fusions to the carboxy-terminus of the target protein. In fact, the initial reports of biotinyl-domain fusion proteins described the construction of carboxy-terminal fusions (14). If the protein requires both free amino- and carboxy-termini, it may be possible to construct the gene fusion such that the biotinyl domain is inserted within the protein coding sequence rather than at the terminal ends. For example, Consler et al. constructed a hybrid polypeptide with the biotinyl-fusion domain inserted internally within a cytoplasmic loop of E. coli lactose permease (43). In summary, we have developed a method to detect binding of 3H-labeled DNA to human topoisomerase I using the scintillation proximity assay. The enzyme is produced in baculovirus-infected insect cells as a chimeric biotinyl-fusion protein. The assay does not require purified protein since the biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts with streptavidin-coated SPA beads. The assay can be readily configured in high-throughput format to facilitate discovery of novel topoisomerase I inhibitors. A similar assay utilizing E. coli topoisomerase I biotinyl-fusion protein has been successfully employed in high-throughput format to identify agents that either (i) inhibit DNA binding or (ii) stabilize a covalent enzyme–DNA intermediate (C. G. Lerner, A. Y. Chiang Saiki, A. C. Mackinnon, and X. Xuei, in press, J. Biomol. Screen.). The technique may also be useful for further characterization of CPT analogs, including kinetic studies of salt-mediated covalent complex reversal. It is likely that the general method of employing biotinyl-fusion protein technology can be applied to other target proteins to facilitate development of novel high-throughput scintillation proximity assays.

ACKNOWLEDGMENTS We thank Tom McGonigal for assistance with baculovirus methodology, Jennifer Fostel for advice on preparation of nuclear extracts, and Leonard Katz for suggesting in vivo labeling as a method for [3H]DNA preparation. We also thank Linus Shen, Jennifer Fostel, Carole Carter, and Leonard Katz for review of the manuscript.

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