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[Cancer Research 59, 3689-3697, August 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 3689-3697, August 1, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

Tethering a Type IB Topoisomerase to a DNA Site by Enzyme Fusion to a Heterologous Site-selective DNA-binding Protein Domain1

Giovanni Luca Beretta, Monica Binaschi, Emanuela Zagni, Luisa Capuani and Giovanni Capranico2

Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan [G. L. B., M. B., E. Z., L. C., G. C.]; and Department of Biochemistry "Moruzzi," University of Bologna, 40126 Bologna [G. C.], Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 Discussion
 REFERENCES
 
Topoisomerase IB (Top1) has essential functions in higher eukaryotes, but effective anticancer agents can transform it into a lethal DNA-cleaving toxin. Fusion of the yeast Gal4 DNA-binding protein domain (amino acids 1–105; Gal4DBD) to the NH2 terminus of full-length human Top1 results in a GalTop chimera that maintains basic properties of the two parent proteins. DNA cleavage and binding activities of GalTop were then compared to Top1 to establish whether the fusion protein had altered site specificity. Under conditions of reduced binding of Top1 to DNA, Gal4DBD was able to selectively anchor the chimera on a template containing a Gal4 consensus motif, thus bringing Top1 to cleave 20–40-bp sequences close to the Gal4 motif with high specificity. Footprinting analyses showed that the chimera protected a DNA region that was wider than that protected by a Gal4DBD protein fragment, consistent with the cleavage results. The data demonstrate that a Top1 can be targeted to a specific DNA site by protein fusion to a heterologous DNA-binding domain. Such hybrid topoisomerase-derived enzymes may be useful for directing Top1 activity to specific genomic loci in living cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 Discussion
 REFERENCES
 
Top13 is a ubiquitous eukaryotic enzyme that governs DNA topology during transcription, replication, recombination, and repair (1) . Top1 is essential in higher organisms, functions as a monomer of a predicted Mr 90,649, and relaxes supercoils by coupling DNA breaking/rejoining and swivel activities (1, 2, 3) . During the reaction pathway, the enzyme binds to a DNA duplex and then cuts a strand with concomitant formation of a protein-DNA intermediate, whereby a tyrosine residue is covalently linked to the 3' terminus. After strand passage, the enzyme reseals the DNA break and restores the initial duplex molecule. In some instances, Top1 can transfer the protein-held strand segment to an unrelated polynucleotide chain, thus generating recombinant molecules. Crystallographic findings, indeed, suggested a common evolutionary origin of Top1 and tyrosine recombinases from an ancestral strand transferase (3, 4, 5) .

Human Top1 is constituted by an unconserved NH2-terminal domain, a conserved core domain, a short unconserved linker, and a conserved COOH-terminal domain containing the active Tyr723 residue (1 , 6) . Both the central and COOH-terminal domains are responsible for the DNA-binding activity, whereas the NH2-terminal region is likely responsible for other functions, such as nuclear localization, enzyme regulation, and protein-protein interactions (6, 7, 8) . Investigations of DNA binding and cleavage activity have shown that a DNA-bound enzyme protects both strands over a region of 15–19 bases, with the strand cut located in the middle (1 , 9 , 10) . It has also been shown that Top1 binds with higher affinity to supercoiled and bent DNA segments (Refs. 11, 12, 13 and references therein), suggesting that it may recognize DNA structural features. DNA cleavage mediated by Top1 has a low degree of site selectivity, and only loose, short consensus sequences could be derived from randomly collected cleavage sites (14, 15, 16) , which are unlikely to regulate the targeting of the enzyme in nuclear chromatin.

Moreover, Top1 is a promising target for cancer and infectious diseases (1 , 10) . Camptothecin and other agents trap DNA-topoisomerase covalent complexes, resulting in enhanced DNA cleavage that can lead to cell death (10 , 16, 17, 18) . Experimental data support the idea that the drug action transforms an essential enzyme into a DNA-damaging cellular toxin (10 , 16 , 17 , 19) . Recently, abasic sites and other lesions have been shown to trap DNA-Top1 complexes, acting as endogenous factors that are able to poison the enzyme (20) . In addition, some amino acid mutations have been shown to confer a lethal phenotype to yeast Top1, likely due to increased levels of DNA damage (21) . Current knowledge, thus, supports the view that, under certain circumstances, DNA topoisomerases are dangerous to the cell (1 , 10 , 17 , 22) , raising the possibility of exploiting these enzymes as efficient cellular toxins.

With the long-term goal of designing a Top1-derived nuclear toxin (17) , we have here asked the question of whether human Top1 can be targeted to a specific DNA site by fusion to a heterologous protein domain. Targeting of a DNA-damaging protein to a specific sequence may be useful for designing a cell-selective toxin. Thus, we have constructed and purified a full-length human Top1 fused to the first 105 amino acids of the yeast transcriptional activator Gal4 protein, corresponding to the site-specific DNA binding and dimerization domains. Our results demonstrate that Top1 can be engineered to selectively target a DNA site.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 Discussion
 REFERENCES
 
Plasmids, Yeast Strains, and Other Materials.
Plasmids pEMBLyex4 and YepG525 (23) were kindly provided by Prof. E. Martegani (Milan University, Milan, Italy). Plasmid pGex-2T was purchased from Pharmacia (Uppsala, Sweden). Plasmid ptac-hTop1 was a generous gift of Prof. J. Wang (Harvard University, Cambridge, MA). Dr. P. Benedetti (Consiglio Nazionale delle Richerche, Rome, Italy) kindly provided Saccharomyces cerevisiae yeast strains JCW1 (MATa, his4-539, lys2-801, ura3-52, top1::HIS4) JN134top1-1 (MAT{alpha}, rad52::LEU2, trp1, ade2-1, his7, ura3-52, ise1, top1-1) and JEL1-{Delta}top1 (MAT{alpha} leu2 trp1,ura3-52, prb1-1122, pep4-3, Dhis3::PGAL10-GAL, {Delta}top1; Refs. 24 and 25 ). A specific monoclonal antibody against Top1 (26) was kindly provided by Dr. Igor Bronstein (York University, York, United Kingdom). A specific Gal4DBD monoclonal antibody was from Clontech (Palo Alto, CA). Camptothecin was purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in DMSO. Agarose and acrylamide were purchased from Bethesda Research Laboratories (Basel, Switzerland). The ECL kit, [{gamma}-32P]ATP, and [{alpha}-32P]dCTP were purchased from Amersham (Milan, Italy). Other plasmids, enzymes, and restriction endonucleases were from New England Biolabs (Taunus, Germany).

Construction of pGLB21 and pEZ-2ThTop1 Plasmids.
The plasmid pEMBLyex4 contains a hybrid GAL-CYC galactose-inducible promoter upstream to a polylinker sequence with unique restriction sites (23) . A DNA fragment coding for the Schistosoma japonicum GST protein was amplified by PCR from pGex-2T and inserted into the polylinker of pEMBLyex4, resulting in pEZ-2T plasmid that had a unique BamHI site at the 3' end of the GST coding sequence. The cDNA and a 3'-untranslated region of human Top1 (3340 bp) was obtained from digestion of the ptac-hTop1 plasmid with BamHI and SalI and was then inserted into the BamHI site of pEZ-2T. In the pEZ2ThTop1 plasmid, the Mr 26,000 GST domain was linked to the NH2 terminus of the human Top1 with an intervening heptapeptide (LVPRGSD).

A DNA fragment coding for the first 105 amino acids of the yeast Gal4 protein was PCR-amplified from plasmid YepG525 and cloned into the BamHI site of pEZ-2ThTop1, and the Gal4-coding region was fully sequenced. The resulting plasmid, pEZ-20, expressed, at low levels, a GST-GalTop fusion protein that could not be purified by affinity chromatography because the GST domain did not bind to the glutathione resin. Moreover, the Gal4DBD domain did not bind to an oligonucleotide containing a Gal4 consensus motif (data not shown), suggesting that the GST and Gal4 domains could interfere with each other. Thus, the pEZ-20 DNA was digested with SacI and PstI to remove the GST coding fragment and transformed into yeast JCW1 cells to allow plasmid ligation in vivo. Plasmid pGLB21 was recovered and shown to have Gal4DBD, Top1, and promoter sequences unmodified. Upon galactose induction, the pGLB21 plasmid expressed high levels of GalTop protein chimera that had an intervening tripeptide (GSD) between Gal4DBD and Top1 sequences. All plasmid DNAs were checked by restriction analyses and by sequencing the insert/vector junctions.

Purification of GalTop, Top1, and Gal4(1-105) Fragment Proteins.
Yeast JEL1 cells transformed with pGLB21 or pEZ2ThTop1 plasmids were galactose-induced, as described (2 , 25 , 27) . Cells were collected by centrifugation and resuspended in PBS buffer [(140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 (pH 7.3)] for pEZ2ThTop1 and TEEG buffer [50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, and 10% glycerol] for pGLB21 plasmids. Protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) were included in all buffers during purification. Cells were then lysed with glass beads by vortexing vigorously, and proteins were fractionated (50 and 70%) with ammonium sulfate precipitation. (a) Top1 was resuspended in PBS, loaded onto a GSH-Sepharose 4B (Pharmacia Biotech) chromatography column, and allowed to bind to the resin. The column was washed with PBS several times, and Top1 was then eluted with 10 mM glutathione in 50 mM Tris-HCl (pH 8.0). Top1 was finally concentrated by a phosphocellulose column and stored in 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.1 mM DTT, 1 M KCl, 200 µg/ml BSA, and 30% glycerol at -80°C. (b) GalTop was resuspended in TEEG, loaded onto a phosphocellulose column, and eluted with a 0.2–1 M KCl linear gradient. Gal4DBD protein was stored in 50 mM Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM EGTA, 0.1 mM DTT, 150 mM KCl, 1 mM phenylmethylsulfonyl fluoride, and 30% glycerol at -80°C. (c) The phosphocellulose procedure was used to purify Top1 as well. Endogenous Gal was not copurified at detectable levels with the phosphocellulose protocol, as shown by Western analyses of purified Top1 (data not shown).

A PCR-amplified DNA fragment coding for the first 105 amino acids of yeast Gal4 protein was cloned into BamHI site of pGex-2T, which results in a GST-Gal4(1-105) fusion protein with a thrombin-specific cleavage site between the two protein partners. Escherichia coli cells transformed with the plasmid were grown in 100 µg/ml ampicillin at 37°C to 0.7 A595 and induced with 0.1 mM isopropyl-ß-thiogalactoside for 3 h. Cells were then sonicated in TEEG buffer containing 10 µM ZnCl2 and protease inhibitors. Proteins were fractionated by ammonium sulfate precipitation at 35 and 65%. GST-Gal4(1–105) protein was loaded onto a heparin-Sepharose column and eluted with a linear 0.15–1.5 M KCl gradient in TEEG buffer with 10 µM ZnCl2. The protein was stored in 20 mM HEPES (pH 7.5), 150 mM NaCl, 1.4 mM 2-mercaptoethanol, and 10% glycerol with 20 µM ZnSO4 at -80°C. The purified Gal4(1–105) fragment was obtained by thrombin digestion in the same buffer before use, and proteolytic digestion of the protein was checked by Western blots.

Band-shifting Assay.
DNA binding reactions (40 µl) were in 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 200 mM KCl, 0.1 µM ZnCl2, 10 µg/ml BSA, and 5% glycerol and contained 32P-labeled wt "Gal4 oligomer" (5'-CGGAGGACTGTCCTCCG; Ref. 28 and purified proteins. Competition experiments were performed adding to the reaction different amounts of cold wt or mutated (5'-CtGAGGACTGTCCTCaG) Gal4 oligomer. The two base mutations (in lowercase letters) are known to reduce the specific binding of Gal4 to its target site (29) . DNA/protein complexes were detected by 4% neutral polyacrylamide gel electrophoresis at 5 V/cm and 4°C in 12.5 mM Tris-HCl (pH 7.5), 95 mM glycine, and 0.5 mM EDTA. Gels were then dried, and fractions of protein-bound 32P-labeled DNA were determined by PhosphorImager analyses. For Southwestern analyses of protein complexes, purified proteins were incubated with unlabeled wt Gal4 oligomer and fractionated in a gel as described for labeled oligomers.

Western and Southwestern Analyses.
Purified proteins were fractionated with 7.5% PAGE (30) . Protein transfer to nitrocellulose membranes was performed in 10 mM 3-cyclohexylamino-1-propanesulfonic acid(pH 11), 0.1 mM methylthioglycolate, and 10% methanol at 4°C for 1 h at 800 mA and then 3 h at 300 mA. Membranes were incubated in 50 mM Tris-HC, (pH 7.5), 400 mM NaCl, 0.1% Tween 20, and 0.5% gelatin for 1 h at 25°C. Monoclonal antibody against human Top1 (26) or Gal4DBD was then added for a further 1 h at 25°C, followed by a second antibody for 1 h at 25°C. Immunocomplexes were then detected with Amersham ECL kit following supplier instructions.

DNA Relaxation and Cleavage Assays.
DNA relaxation activity was assayed with 250 ng of supercoiled pBR322 DNA in 20 mM Tris-HCl (pH 7.5), 0.1 mM DTT, 5 mM MgCl2, 0.5 µg/ml BSA, 0.1 mM EDTA, and from 100 to 300 mM KCl for 0.5 h at 37°C (27) . For cleavage experiments, a single copy of the wt Gal4 oligomer was cloned into a plasmid DNA. Then, a plasmid DNA fragment containing the Gal4 motif was 3'-end 32P-labeled, as described (15 , 27) and incubated with purified proteins in 30 µl of 20 mM Tris-HCl (pH 8), 0.1 mM DTT, 5 mM MgCl2, 1 µg/ml BSA, 0.1 µM ZnCl2, and 50 µM camptothecin and different KCl concentrations at 37°C for 20 min. Reactions were stopped by 1% SDS and 0.3 mg/ml proteinase K at 42°C for 45 min. DNA breaks were then analyzed by 8% polyacrylamide denaturing gels. Cleavage levels were measured by PhosphorImager analyses.

DNA Footprinting Assay.
GalTop binding to Gal4 consensus motif was assayed by DNase I protection assay. A 32P-labeled DNA fragment containing a Gal4 motif, which was used in the cleavage experiments, was also used for footprinting analyses. Binding reaction (30 µl) was in 20 mM Tris-HCl (pH 8), 5 mM MgCl2, 10 µM ZnCl2, 0.1 mM DTT, 1 µg/ml BSA, 10 µg/ml salmon sperm DNA, and 50 or 200 mM KCl for 20 min at 25°C. Then, DNase I (Sigma) was added at 6.6 units/ml for 1 min and then stopped by 10 mM EDTA and 0.3 mg/ml proteinase K. DNA fragments were finally examined by sequencing gels and PhosphorImager analyses, as described above.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
Discussion
 REFERENCES
 
Functional Properties of GalTop Protein Chimera.
Fusion of the DNA-binding domain (amino acids 1–105) of Gal4 (Gal4DBD) to the NH2 terminus of human Top1 yielded a protein chimera (GalTop) that retained the functions of the components (Fig. 1)Citation . Proteins were overexpressed in yeast, and to allow purification near homogeneity by affinity chromatography, we fused Top1 to a GST domain (Fig. 1A)Citation . However, the same GST domain interfered with Gal4DBD activity when fused to it (data not shown). Thus, the GST cDNA was removed from the yeast expression vector to yield the GalTop chimera (Fig. 1ACitation ; see "Materials and Methods" for other details). Immunoblot analyses showed that both GalTop and Top1 were recognized by a specific monoclonal antibody against Top1, whereas a specific anti-Gal4DBD antibody recognized GalTop only (Fig. 1B)Citation . Proteolytic fragments were detected in some preparations of purified proteins. In particular, a fragment of Mr ~45,000 was detected in some GalTop preparations by the antiTop1 antibody only (Fig. 1B)Citation . The molecular mass was much lower than that of the Mr 68,000 fragment commonly detected in purified topoisomerase I preparations (2 , 6 , 10) . The antiTop1 used antibody recognized an epitope in the central DNA-binding domain of Top1,4 therefore, the Mr 45,000 fragment likely corresponded to a portion of enzyme central domain. Because proteolytic fragments of Top1 can reconstitute in vitro, showing relaxation activity at 50–200 mM salt but no activity above 250 mM salt (31) , we tested our purified GalTop for relaxation activity at 100 and 300 mM KCl and 5 mM MgCl2 in comparison with Top1. Our purified GalTop chimera was able to relax negatively supercoiled plasmids at 100 mM (data not shown) as well as at 300 mM salt (Fig. 1BCitation , bottom), excluding the possibility that a reconstituted protein was responsible for DNA relaxation activity. Thus, the results demonstrate that human Top1 remains functional when fused to Gal4DBD.



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  		Fig. 1. A, diagram of Top1 and GalTop fusion proteins. *, the active Tyr723 at the COOH terminus of Top1 ({square}). GST () indicates the first 220 amino acids of S. japonicum GSH-transferase enzyme. Gal4(1–105) () indicates the first 105 amino acids of yeast Gal4 protein. B, biochemical characterization of GalTop and Top1. The proteins were overexpressed in yeast and purified as described in "Materials and Methods." Top, immunoreaction of Top1 (10 units; Lanes 1 and 3) and GalTop (20 units; Lanes 2 and 4) with specific anti-Top1 and anti-Gal4DBD monoclonal antibodies, following a 7.5% polyacrylamide gel electrophoresis. Bottom, relaxation of supercoiled plasmid pBR322 DNA was assayed in 20 mM Tris-HCl (pH 7.5), 300 mM KCl, 0.1 mM DTT, 5 mM MgCl2, 0.5 µg/ml BSA, and 0.1 mM EDTA and analyzed by 0.7% agarose gels, followed by ethidium bromide staining. Lane 1, supercoiled DNA; Lanes 2–5 and 6–9, reactions with 4, 2, 1, and 0.5 units of Top1 and GalTop, respectively. S and R, supercoiled and relaxed DNAs, respectively.

 
Strand Cleavage by GalTop in a Gal4 Motif-containing DNA Fragment.
Top1-dependent DNA cleavage was then investigated by sequencing gels to establish whether the Gal4DBD-fused Top1 domain was selectively directed to Gal4 DNA sites. A 17-bp oligomer corresponding to a Gal4 DNA consensus motif (5'-CGGAGGACTGTCCTCCG; Ref. 28 ) was cloned into a plasmid DNA. Then, a plasmid fragment containing the motif was uniquely 3'-end32P-labeled and used as substrate in the cleavage assay. DNA cleavage was enhanced by 50 µM camptothecin, a specific Top1 poison, and GalTop was compared to Top1 at similar levels of overall cleavage (Fig. 2)Citation . First, Top1 and GalTop promoted DNA cleavage at identical sites at 100 mM KCl, and enzyme sensitivity to camptothecin was identical (Fig. 2)Citation . Moreover, strand breaks were reversible upon addition of 0.6 M KCl for both proteins (data not shown). Thus, DNA cleavage activity of Top1 was not altered by the fusion to Gal4DBD.



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  		Fig. 2. Targeting of GalTop-mediated DNA cleavage to sequences near a consensus Gal4 DNA motif. A DNA fragment containing one Gal4 consensus motif () was uniquely 3'-end 32P-labeled and incubated with 2 units of Top1 or GalTop in 20 mM Tris-HCl (pH 8), 0.1 mM DTT, 5 mM MgCl2, 1 µg/ml BSA, 0.1 µM ZnCl2, 50 µM campthotecin, and KCl for 20 min at 37°C. Reactions were stopped by 1% SDS and 0.3 mg/ml proteinase K. DNA was then run through 8% sequencing gels. Left and right, upper and lower strands, respectively. Lanes m, purine markers; Lanes c, control DNA; Lanes e, reaction at 100 mM KCl without campthotecin. Triangles, KCl at 100, 200, 240, 270, and 300 mM, from right to left. Arrows, cleaved fragments. Arrowhead, cleavage site near the center of the Gal4 motif.

 
Then, we asked whether cleavage sites were altered under conditions of reduced DNA binding affinity of Top1 but not of Gal4DBD (2 , 32) . Thus, cleavage was assayed at KCl concentrations in the range of 100–300 mM, and the results showed that cleavage levels were higher at 200–240 than 100 mM KCl for GalTop but not for Top1, at sites close to the Gal4 motif (Fig. 2)Citation . At higher concentrations (270–300 mM), cleavage was abolished in the case of Top1, whereas it was still high in the case of the chimera (Fig. 2)Citation . Interestingly, GalTop-mediated cleavage was less persistent at more distant sites (Fig. 2Citation , left bottom; see also Fig. 3Citation ) and was absent at a site mapped within the Gal4 consensus motif (Fig. 2Citation , arrowhead). We must note that GalTop promoted DNA breaks on either side of the motif and in both the two strands (Fig. 2)Citation . Therefore, the fused Gal4DBD could compensate the reduced DNA binding affinity of Top1 in a Gal4 motif-containing substrate.



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  		Fig. 3. EDTA (left) and Gal4(1–105) protein fragment (right) inhibit GalTop-mediated DNA cleavage at high salt. A 3'-end-labeled DNA fragment containing one Gal4 motif () was used as substrate. Left, DNA was incubated with GalTop (2 units) in 20 mM Tris-HCl, (pH 8), 0.1 mM DTT, 1 µg/ml BSA, 50 µM campthotecin, and 100 or 270 mM KCl at 37°C for 20 min. Reactions were then processed as described in the legend to Fig. 2Citation . Triangles, EDTA at 0, 2, 5, 10, and 20 mM, from right to left. Arrows, cleaved fragments. Lane m, purine markers; Lane c, control DNA; Lane z, no EDTA and 0.1 µM ZnCl2. Right, reactions were in 20 mM Tris-HCl, (pH 8), 0.1 mM DTT, 1 µg/ml BSA, 5 mM MgCl2, 0.1 µM ZnCl2, 270 mM KCl, and 50 µM campthotecin and stopped as above. Lane m, purine markers; Lane c, control DNA; Lanes 1 and 2, Top1 (2 units); Lanes 3 and 4, GalTop (2 units); Lanes 1 and 3, 2.1 µg of Gal4(1–105); Lanes 2 and 4, 3.5 µg of Gal4(1–105). *, contaminating bands.

 
To investigate whether this was due to Gal4DBD fusion, we performed cleavage reactions in the presence of EDTA, a metal ion-chelating agent, and in the absence of Zn2+ (an essential cofactor of Gal4DBD; Ref. 33 ). GalTop-mediated DNA cleavage was abolished under these conditions at 270 mM KCl, whereas it was completely unaffected at 100 mM KCl (Fig. 3Citation , left). Moreover, because Top1 has a higher binding affinity for bent DNA segments (11) , we tested whether Gal4-caused DNA bending (33) was responsible for salt-persistent cleavage of Top1 at sites close to the motif. DNA cleavage promoted by GalTop or Top1 was assayed in the presence of a Gal4(1–105) protein fragment at 270 mM KCl (Fig. 3Citation , right). The amounts of the Gal4(1–105) fragment added to the reactions were comparable to those that were shown to bind to the motif by footprinting analyses (see below). The Gal4 fragment had no effect on the cleavage activity of Top1, whereas it completely suppressed DNA cleavage by GalTop (Fig. 3)Citation . The latter effect was likely due to competition between Gal4(1–105) protein fragment and GalTop chimera for binding to the Gal4 motif. Thus, the findings demonstrated that only a Top1-fused Gal4DBD could determine an efficient DNA binding to and cleavage of the substrate by Top1 at 240–270 mM KCl.

Targeting of Cleavage to Sequences Nearby a Gal4 Consensus Motif.
The above results demonstrated that 240–270 mM KCl prevented formation of Top1-dependent DNA breaks in the case of Top1 but not for the chimera. Thus, we tested the idea that the Gal4DBD binds to its specific sequence motif and, in so doing, brings the Top1 domain at sites close to the Gal4 sequence. DNA cleavage induced by the chimera was then investigated in a DNA fragment containing the Gal4 motif and in an unrelated fragment without Gal4 sequences (Fig. 4)Citation . Again, at 100 mM KCl, cleavage patterns were identical for Top1 and GalTop in both DNA fragments. Interestingly, DNA cleavage activity of the chimera was markedly changed as compared to Top1 at 240–270 mM in the Gal4 motif-containing fragment but not in the one lacking the motif (Fig. 4Citation ). In the latter, Top1 as well as GalTop produced few or no breaks at 240–300 mM KCl. In the former, the response of cleavage sites to salt increase was strikingly dependent on the distance of the site from the 17-bp motif. At 240–270 mM KCl, the chimera-dependent DNA breaks close to the Gal4 motif were present, whereas those distant from the Gal4 motif were progressively decreased (Fig. 4Citation , left).



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  		Fig. 4. Influence of the distance from a Gal4 DNA motif on GalTop-mediated DNA cleavage at high salt. A DNA fragment containing a Gal4 motif (, left) and an unrelated DNA fragment with no Gal4 motif were 3'-end- or 5'-end-labeled, respectively, and then reacted with the indicated proteins (2 units). Reaction conditions and lanes are as described in the legend to Fig. 2Citation . * and arrows, contaminating fragment and cleavage products, respectively.

 
Cleavage levels were determined by PhosphorImager analyses, and the ratios of the levels at 270 mM over those at 100 mM KCl were plotted as a function of the distance of the site from Gal4 motif (Fig. 5)Citation . At sites <=70 bp distant from the motif, cleavage was high at 270 mM, with some sites at 30–35 bp from the motif showing prominent cleavage levels. In contrast, cleavage was markedly reduced at more distant sites or in fragments without Gal4 motifs (Fig. 5)Citation . Thus, GalTop-mediated DNA breaks were restricted to sites close to the Gal4 motif under high salt conditions.



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  		Fig. 5. Cleavage is targeted close to a Gal4 motif. The ratio of the cleavage level at 270 mM over that at 100 mM KCl is plotted against the distance of the site from the central base pair of the Gal4 DNA motif (). {square} and {blacksquare}, GalTop and Top1, respectively. {omega}, sites of DNA fragments without Gal4 sequence motifs. Columns, means of two to three independent determinations; SEs were 15% of mean.

 
Specific Binding of GalTop Chimera to a Gal4 DNA Motif.
We directly examined the specificity of the DNA binding activity of GalTop by a band-shifting assay (Fig. 6)Citation . GalTop but not Top1 could bind to a 17-bp oligomer corresponding to a consensus Gal4 DNA sequence motif (see above), and two protein-DNA complexes were observed (Fig. 6Citation , top). The protein-binding assay was also performed with cold oligomers, and after a native gel run, DNA-protein complexes were examined by Southwestern analyses. Specific antibodies against either Top1 or Gal4DBD could recognize the protein-DNA complexes (Fig. 6)Citation , thus showing that the chimera was oligomer-bound and likely responsible for the observed band shifts. However, we do not know the precise nature of the two complexes.



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  		Fig. 6. Specific binding of GalTop to a Gal4 consensus motif. A double-stranded 17-bp DNA oligomer corresponding to a Gal4 consensus motif (see "Materials and Methods") was incubated with 10 units of GalTop (Lanes 2) or Top1 (Lanes 3) in 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 200 mM KCl, 0.1 µM ZnCl2, 10 µg/ml BSA, and 5% glycerol at 25°C for 20 min. Samples were then fractionated on a 4% nondenaturing polyacrylamide gel. Lanes 1, control DNA. *, contaminating band. Top left, the substrate was 32P-labeled. Top right, cold oligomers were used as substrate; DNA/protein complexes (arrows) were detected by Southwestern analyses. Bottom, equilibrium competition DNA binding assay with cold wild-type (•) or double mutant ({blacksquare}) oligomers as competitor DNA (see "Materials and Methods"). Data points, percentages of 32P-labeled DNA in protein/DNA complexes, plotted against the concentration of competitor oligomers.

 
The specificity of DNA binding by the chimera was then tested by an equilibrium competition assay. The palindromic CGG triplets at the ends of the Gal4 17-bp motif are crucial for selective binding of the Gal4 protein (29) . Thus, a labeled wt oligomer was mixed with different concentrations of unlabeled wt oligomer or an oligomer bearing mutated triplets (CtG) at its ends. These mutations markedly reduced the affinity of Gal4 for the sequence motif (29) . The results showed that the wt oligomer was ~-10-fold more potent than the mutated one in reducing the binding of the chimera to the labeled DNA (Fig. 6Citation , bottom). Thus, the Gal4DBD allowed the chimera to specifically anchor itself on a template containing a Gal4 consensus motif.

The Binding Site of GalTop is Wider Than Gal4 Consensus Sequence.
Next, we determined the precise binding site of the chimera by DNase I footprinting analyses (Fig. 7)Citation . Footprinting of the Gal4(1–105) protein fragment was also examined, and the results confirmed the specific binding of the Gal4(1–105) domain to the consensus Gal4 motif (Fig. 7Citation , left; Ref. 34 ), which was used in the cleavage and band-shifting assays (see above). In agreement with Gal4(1–105), GalTop protected the 17-bp sequence motif at 200 mM KCl (Fig. 7)Citation . At lower salt concentrations, a site-specific protection was not detected, likely due to stronger DNA binding affinity of the topoisomerase domain (data not shown). Consistently, Top1 showed a uniform and progressively higher protection along the entire DNA fragment at any salt concentration tested (Fig. 7Citation and data not shown).



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  		Fig. 7. Footprinting analysis of the binding site of GalTop. A DNA fragment, containing one Gal4 sequence motif (), was uniquely 5'-end-labeled and incubated with purified proteins in 20 mM Tris-HCl (pH 8), 0.1 mM DTT, 1 µg/ml BSA, 5 mM MgCl2, and 10 µM ZnCl2. Then, samples were subjected to limited DNase I digestion (6.6 units/ml) and analyzed with 8% polyacrylamide sequencing gels. A, DNase I protection by Gal4(1–105) protein fragment (0.7, 3.5, and 7 µg) at 50 mM KCl. B, footprinting of Top1 (0.5, 2, and 6 units) and Gal4-Top1 (1, 6, and 20 units) at 200 mM KCl. Lanes m, purine markers; Lanes c, undigested DNA; Lanes d, DNase I fragmentation of naked DNA fragments. {circ}, sites of DNase I enhancements; braces, additional regions of protection by GalTop.

 
Interestingly, DNase I protection by the chimera was also detected in two other regions of ~10–12 bp (Fig. 7Citation , braces) at either side of the motif that were not protected by the Gal4(1–105) fragment. In addition, two sites of enhanced DNase I degradation in the presence of the Gal4(1–105) fragment were not observed in the case of the chimera (Fig. 7Citation , open circles). These observations, thus, indicated that the chimera protected a wider region than Gal4(1–105) protein fragment extending on either side of the Gal4 consensus motif. Because the chimera likely binds to the DNA as a dimer, the observed protection is likely due to three DNA-binding protein domains: one Gal4DBD and two independent Top1 domains. The Gal4DBD fusion can, thus, bring two Top1 proteins to bind to sequences nearby a Gal4 consensus sequence (Fig. 8)Citation , in very good agreement with the cleavage data.



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  		Fig. 8. A working model for DNA binding/cleavage mode of the GalTop chimera. Gal4DBD anchors the entire protein to the DNA template by binding to its specific sequence motif. Top1 may, thus, bind to and cleave nearby DNA sequences. Because both strands are cut by the chimera, Top1 may be oriented relative to the Gal4DBD dimer in either one of two ways (compare the left Top1 in A and B). OH, the free 5' terminus at the strand cut, shown for the left Top1 only. Top1 structure is shown as an asymmetric object (3 , 4) .

 

   Discussion
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 Discussion
REFERENCES
 
These results demonstrate that the full-length human Top1 can be targeted to a Gal4 motif by protein fusion to the DNA-binding domain of yeast Gal4 protein. Thus, a fused heterologous protein domain can change, under certain conditions, the site selectivity of DNA binding and cleavage activity of a Top1. Type IB DNA topoisomerases play essential roles in several nuclear processes, but they can be transformed into DNA-damaging cellular toxins by the action of antitumor agents or by common nucleotide alterations (10 , 16 , 17 , 22) . The targeting of enzyme activity to a specific site may be of high value in the further elucidation of enzyme functions as well as in the long-term goal of designing a site-specific protein DNA cutter with enhanced lethal potential against tumor cells or microbial pathogens.

Footprinting analyses show that the GalTop chimera protects a Gal4 motif as well as two other regions of 10–12 bp at either side of the motif. The wider protection is in very good agreement with the cleavage data and may be due to the three separated DNA-binding domains of the chimeric dimer. The Gal4 domains of two chimera molecules likely dimerize and bind to the Gal4 consensus motif, thus bringing two Top1 domains to contact the DNA on either side of the motif (Fig. 8)Citation . The crystal structure of human Top1 has provided important information for understanding the catalytic mechanism of the enzyme (3 , 4) . The authors have proposed that DNA relaxation occurs via a "controlled rotation" mechanism, suggesting that swivel of DNA strands is not free but is somewhat steered by the protein (4) . These studies show that human Top1 is asymmetric, indicating that Top1 can bind to a double helix in two possible orientations (Fig. 8)Citation . These two possibilities can be distinguished by determining the strand that is cut by the protein, because the cleavable strand is determined by the orientation of Top1 bound to the DNA (3 , 4) . Because we have observed that both strands can be cut by GalTop at both sides of the Gal4 motif, we may infer that the two Top1 domains of the chimera can still bind in either orientation to sequences close to the motif (Fig. 8)Citation , suggesting a certain degree of protein flexibility. However, for one of these two possibilities, the DNA-bound Gal4DBD would likely impede the rotation of the broken strand around the uncut one (Fig. 8Citation , top left). Whether the relative conformations of Top1 and Gal4 domains of the chimera have an effect on the relaxation activity of Top1 remains to be established by further investigation. We must say that the model depicted in Fig. 8Citation is speculative because the intervening DNA between a Top1 domain and the Gal4DBD might be looped in the case of cleavage sites relatively distant from the Gal4 consensus. In addition, our footprinting data cannot rule out the possibility that one half only of a GalTop dimer can bind to DNA at any given time.

Top1-DNA interactions are unlikely to determine the sites of enzyme activity in nuclear chromatin (1 , 2) . The NH2-terminal domain, which has not been conserved during evolution, has been recently shown to be responsible for localization of Drosophila Top1 to transcriptionally active chromatin domains by interacting with transcription protein complexes (7) . Top1 can, indeed, function as a transcriptional coactivator or repressor of basal transcription in in vitro systems by forming an active TFIID-TFIIA initiation complex (8 , 35 , 36) . In addition, biochemical results provided evidence that the NH2-terminal domain of human Top1 can also interact with nucleolin, a common nucleolar protein, and with Simian virus 40 large T antigen (37, 38, 39) . Thus, the NH2-terminal domain, which is dispensable for the catalytic reaction of Top1, can play a pivotal role in determining the localization of Top1 in nuclear chromatin by contacting other nuclear proteins. Our results show that the GalTop chimera may mimic the physiological tethering of Top1 to genomic sites by proteins such as TFIID (8) . On the basis of this knowledge, we may predict that GalTop, which maintains the Top1 NH2-terminal domain, would continue to respond to cellular programs of gene expression during cell cycle. This would likely interfere with an efficient in vivo targeting of the chimera to Gal4 DNA motifs. Nevertheless, the general approach of targeting DNA topoisomerases to unique genomic sites by protein fusion may be fruitfully used to further understand the cellular functions of the enzymes.

Hybrid DNA-binding proteins have been constructed to generate DNA-cleaving enzymes with novel sequence selectivity. Fusion to heterologous DNA-binding domains has been shown to change the sequence specificity of the restriction endonuclease FokI (40 , 41) , HIV integrase (42, 43, 44) , and T7 RNA polymerase (45) . FokI consists of two functionally distinct domains: one for the recognition of the binding site and the other for the endonucleolytic activity (40 , 41) . The substitution of the first one with an unrelated site-specific DNA-binding domain produced a chimeric FokI enzyme that has the site selectivity of the new recognition domain (40 , 41) . Our approach in engineering human Top1 has been different since a new domain was added to the full-length enzyme, similarly to HIV integrase and T7 RNA polymerase. The full-length HIV integrase has been fused to the DNA binding domain of {lambda} repressor, E. coli LexA protein, or zinc finger protein zif268 (42, 43, 44) . In all cases, hybrid integrases preferentially targeted in vitro and in vivo integration to sites near the motif recognized by the fused heterologous protein domain (42, 43, 44) . These studies have established that several combinations of integrase/binding domains can be functional. Similarly, T7 RNA polymerase is functional when fused to a Gal4 protein (45) . Because both HIV integrase and Top1 have a poorly site-selective activity, the results show that a highly site-selective binding domain may efficiently modulate DNA site selection by less specific DNA-binding proteins. Knowledge of further structural determinants of Top1 binding to DNA will help in engineering Top1 chimera with a higher site selectivity.

In summary, our study demonstrates that tethering a Top1 to a selective DNA sequence can be obtained by enzyme fusion to a heterologous site-selective DNA-binding protein domain. Such hybrid topoisomerase-derived enzymes may direct topoisomerase activity to specific genomic loci in living cells as well and may be useful for further evaluating the molecular mechanism and nuclear functions of these essential enzymes. Moreover, the success in targeting a DNA-damaging protein to a specific sequence gives support to the idea of designing a cell-selective nuclear toxin based on DNA sequence differences among cells.


   ACKNOWLEDGMENTS
 
We are grateful to Stella Tinelli for skillful technical assistance. We wish to thank J. Wang (Harvard University, Cambridge, MA), P. Benedetti (Consiglio Nazionale delle Richerche, Rome, Italy), I. Bronstein (York University, York, United Kingdom), and E. Martegani (Milan University, Milan, Italy) for providing us with yeast strains, antibodies, and plasmids.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro, Milan, Italy (to G. C.). Back

2 To whom requests for reprints should be addressed, at Istituto Nazionale per lo Studio e la Cura dei Tumori, via Venezian 1, 20133 Milan, Italy. E-mail: capranico{at}biafarm unibo.it. Back

3 The abbreviations used are: Top1, topoisomerase type IB; Gal4DBD, Gal4 DNA-binding protein domain; GST, glutathione S-transferase; wt, wild-type. Back

4 I. Bronstein, personal communication. Back

Received 3/25/99. Accepted 5/28/99.


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 Discussion
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