Vol. 11, No. 10

MOLECULAR AND CELLULAR BIOLOGY, OCt. 1991, p. 4973-4984 0270-7306/91/104973-12$02.00/0 Copyright © 1991, American Society for Microbiology

Chromatin Structure, Not DNA Sequence Specificity, Is the Primary Determinant of Topoisomerase II Sites of Action In Vivot ANDOR UDVARDYt AND PAUL SCHEDL*

Department of Biology, Princeton University, Princeton, New Jersey 08544 Received 7 March 1991/Accepted 2 July 1991

In the studies reported here we have used topoisomerase II as a model system for analyzing the factors that determine the sites of action for DNA-binding proteins in vivo. To localize topoisomerase II sites in vivo we used an inhibitor of the purified enzyme, the antitumor drug VM-26. This drug stablizes an intermediate in the catalytic cycle, the cleavable complex, and substantially stimulates DNA cleavage by topoisomerase II. We show that lysis of VM-26 treated tissue culture cells with sodium dodecyl sulfate induces highly specific double-strand breaks in genomic DNA, and we present evidence indicating that these double-strand breaks are generated by topoisomerase II. Using indirect end labeling to map the cleavage products, we have examined the in vivo sites of action of topoisomerase II in the 87A7 heat shock locus, the histone repeat, and a tRNA gene cluster at 9OBC. Our analysis reveals that chromatin structure, not sequence specificity, is the primary determinant in topoisomerase H site selection in vivo. We suggest that chromatin organization may provide a general mechanism for generating specificity in a wide range of DNA-protein interactions in vivo.

Additional insights into how topoisomerase II interacts with DNA have come from studies on the substrate properties of DNA segments containing high or low densities of strong cleavage sites (19). In competition filter-binding assays with restriction fragments containing a high (e.g., the 87A7 intergenic spacer) and a low (e.g., the hsp7O gene) density of cleavage sites, the high-density fragment was found to be preferentially retained by topoisomerase II (>10 fold at 50 mM salt). A comparison of dissociation rates indicates that the difference in binding is largely due to the much greater kinetic stability of topoisomerase II-DNA complexes formed with fragments containing a high density of strong cleavage sites. The relaxation of supercoiled plasmids is also influenced by the density of strong cleavage sites, and it appears likely that the high-affinity binding/ strong cleavage sites correspond to the preferred sites for the catalysis of strand passage events (19). The sequence specificity of purified topoisomerase II in DNA cleavage, binding, and catalysis suggests that interactions with specific sites or DNA segments may be important in the functioning of the enzyme in vivo. As an initial attempt to define the sites of action for topoisomerase II, we examined the pattern of double-strand breaks induced by the endogenous enzyme in nuclei prepared from Drosophila tissue culture cells (22). Since cleavage by the endogenous nuclear enzyme is too inefficient to detect, we used the antitumor drug VM-26 (12). Studies on purified topoisomerase II indicate that VM-26 stabilizes the cleavable complex and stimulates DNA cleavage (2, 22). It does not, however, appear to greatly perturb the site specificity of the Drosophila enzyme, and we found that the distribution and relative yield of different cleavage products (at the level of resolution provided by agarose gels) is quite similar to that observed without the drug (22). We found that VM-26 also stimulated DNA cleavage in isolated Drosophila nuclei, and our analysis of the parameters of the cleavage reaction indicated that the double-strand breaks were generated by the endogenous topoisomerase II. In the work reported here we have used VM-26 to examine the sites of action for topoisomerase II in vivo in Drosophila KC cells.

Although the higher-order structure of eukaryotic chromosomes has been the focus of considerable attention, very little is known about the factors that determine the longrange organization of the DNA-chromatin fiber. One enzyme which is likely to have some role in higher-order structure is topoisomerase II. In vitro topoisomerase II catalyzes a variety of alterations in the topological organization of DNA; in vivo it is thought to function in an analogous fashion to govern the topology of the DNA-chromatin fiber. In addition to its catalytic activities, topoisomerase II has been implicated as a structural component of eukaryotic chromosomes (for reviews, see references 5 and 23). The topoisomerization reactions catalyzed by the purified enzyme proceed via a strand breakage-rejoining mechanism involving the transient formation of a covalent DNA-protein complex (8, 11, 17). These transient complexes can be captured by the addition of a protein denaturant, generating a double-strand scission with a topoisomerase II moiety covalently linked to the 5' end of each strand. Analysis of double-strand breaks generated in naked DNA substrates by purified Drosophila topoisomerase II has revealed two factors which may be of some importance in enzyme function. First, topoisomerase II exhibits a relatively high degree of sequence specificity in selection of cleavage sites (18). Second, there is an unusual partitioning of preferred cleavage sites in eukaryotic DNAs: rather than being uniformly distributed along the DNA, the sequences recognized by topoisomerase II are clustered in specific regions (20). For example, in a recombinant containing the two 87A7 hsp7O heat shock genes, most of the major cleavage sites are located in the intergenic spacer separating the genes and at the 5' and 3' boundaries of each transcription unit. In contrast, there are few if any strong cleavage sites within the genes or in the 3'-flanking spacers. * Corresponding author. t This paper is dedicated to the memory of a friend, A. Worcel. t Permanent address: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, 6701 Szeged, Hungary.

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Topoisomerase II cleavage and mapping. For in vivo experiments, Drosophila KC cells (ca. 100 to 200 ml) were treated with different concentrations of VM-26 for 15 to 60 min, harvested by centrifugation, and resuspended in buffer containing 60 mM NaCl, 15 mM Tris-Cl (pH 7.5), and 250 mM sucrose. To induce double-strand breaks, the cells were lysed by the addition of sodium dodecyl sulfate (SDS) to 1%. After a 10-min incubation at room temperature, EDTA (final concentration, 25 mM) and proteinase K (100 ,ug/ml) were added and the lysate was incubated at 65°C for several hours or at 37°C overnight. After three phenol extractions the DNA was ethanol precipitated three times and resuspended in TE (10 mM Tris-Cl [pH 7.5], 1 mM EDTA) for restriction enzyme digestion. Topoisomerase II cleavage in nuclei and in naked DNA was performed as described in reference 22. Chromatin digests of Drosophila KC nuclei were performed essentially as described in reference 21. The topoisomerase II-induced cleavage products were analyzed by indirect end labeling on 0.8 to 1.2% agarose gels that were 40 cm long. Purification of DNA-protein complexes. KC tissue culture cells treated with VM-26 for 15 to 60 min were harvested, and double-strand scissions were induced by lysis with SDS (final concentration, 2.5%) at room temperature. The lysates were incubated (in the presence of 25 mM EDTA and 0.2 mM phenylmethylsulfonyl fluoride [PMSF]) at 65°C for 4 to 5 h to disrupt as completely as possible the noncovalent DNA-protein complexes and to ensure that most cellular proteins were denatured. The DNA-protein complexes were then purified (i) by pelleting overnight at 25,000 rpm through a 8- to 9-ml 25% sucrose column (containing 10 mM Tris-Cl [pH 7.5], 25 mM EDTA, and 0.2 mM phenylmethylsulfonyl fluoride) in an SW41 rotor or (ii) by pelleting overnight at 40,000 rpm through a 10% glycerol-3 M CsCl step gradient in an SW50.1 rotor. The supernatants were discarded, and the pellets were dried and then resuspended in TE containing 0.2 mM phenylmethylsulfonyl fluoride. For restriction enzyme digestion, an aliquot of DNA was incubated with restriction enzyme in the appropriate restriction buffer. After addition of SDS to 1%, the sample was split in two, and one portion was digested with proteinase K (100 jig/ml). The samples were loaded on an agarose gel (buffer and gel containing 0.5% SDS), electrophoresed, and blotted. To analyze the protein in the pellets, we digested aliquots with DNase I (15 ,ug/ml) in 10 mM Tris-10 mM MgCl2 and then concentrated the residual protein by trichloroacetic acid precipitation and washing with acetone. After boiling, the protein was analyzed on a 7% acrylamide gel and electroblotted to nitrocellulose, and the blot was probed with a rabbit polyclonal anti-Drosophila topoisomerase II antibody (a kind gift from M. Sanders and T. Hsieh). RESULTS VM-26 stimulates the formation of double-strand breaks in Drosophila tissue culture cells. To determine whether VM-26 stimulates the formation of double-strand scissions in vivo, we incubated Drosophila KC tissue culture cells with increasing concentrations of the drug. The cells were harvested, resuspended in a small volume, and then split into two equal portions. Since DNA cleavage by purified topoisomerase II (with or without VM-26) requires the addition of a protein denaturant, one portion was lysed directly by the addition of SDS, while the other portion was used as a control (see below). After purifying the cellular DNA, we

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FIG. 1. VM-26-stimulated DNA cleavage in vivo in the 87A7 heat shock locus. Termination of the in vivo cleavage reaction with SDS or EDTA. KC tissue culture cells were treated with increasing concentrations of VM-26 for 30 min at 25°C. The cells were harvested by centrifugation, resuspended in a small volume (3 ml), and divided into two portions. One portion was lysed directly by the addition of SDS to a final concentration of 1%, and EDTA and proteinase K were then added after 5 min of incubation. The other portion was preincubated with 25 mM EDTA and 1% NP-40 and then lysed with SDS and digested with proteinase K. The DNA was isolated from each sample by phenol extraction and ethanol precipitation and then restricted with EcoRI. After electrophoresis and blotting, the filter was probed with an EcoRI-SalI restriction fragment from the proximal end of the 87A7 heat shock locus. This displays the fragment pattern reading across the proximal hsp7O gene, the intergenic spacer, and then the distal gene (see diagram). Lanes: 1, no VM-26; 2, 5 Kg of VM-26 per ml; 3, 10 Kg of VM-26 per ml; 4, 25 Kig of VM-26 per ml; 5, 50 ,ig of VM-26 per ml; 6, 100 ,ug of VM-26 per ml. DNA in lanes m was prepared from micrococcal nuclease digests of isolated KC cell nuclei. EDTA, EDTA/NP-40terminated incubation; SDS, SDS-terminated incubation.

visualized drug-induced double-strand breaks in the 87A7 heat shock locus by indirect end labeling. The DNA was restricted with EcoRI and, after gel electrophoresis and blotting, probed with an EcoRI-SalI fragment derived from the proximal side of 87A7. As indicated by the diagram in Fig. 1, this displays the drug-induced cleavage products reading from the proximal 3' spacer through the proximal hsp7O gene into the intergenic spacer and the distal hsp7O gene. For reference, the micrococcal nuclease cleavage pattern in KC cell chromatin is also shown.

VOL . 1 l, 1991

As can be seen in the autoradiograph, VM-26 stimulates the formation of double-strand breaks in the 87A7 heat shock locus in vivo. Although few if any fragments can be detected in the DNA sample isolated from KC cells incubated without VM-26 (lane 1), a series of discrete although weakly labeled cleavage products are observed even at the lowest level of the drug (lane 2). As the drug concentration is increased (lanes 3 to 6), there is a concomitant stimulation of the cleavage reaction until it appears to plateau at high levels of the drug. The most prominent cleavage products are derived from the intergenic spacer and from sequences near the 5' end of each hsp7O transcription unit. There are also some minor cleavage products from within each gene and from the 3'-flanking spacers. Although the relative stimulation of the cleavage reaction appears to depend on the amount of VM-26, we can detect no obvious differences in site specificity between the lowest and highest levels of the drug. In fact, all of the cleavage products observed in cells treated with the higher concentrations of VM-26 can also be found at lower levels when the autoradiograph is overexposed (data not shown). Parameters of the VM-26-stimulated cleavage reaction. To ascertain whether topoisomerase II is responsible for the formation of double-strand scissions in VM-26-treated cells, we examined some of the parameters of the in vivo cleavage reaction. (i) SDS versus EDTA. In the absence of VM-26, the cleavable complex formed by purified Drosophila topoisomerase II can be reversed by the addition of EDTA, and few if any double-strand scissions are observed when this chelator is added prior to SDS. Although previous studies (22) have shown that the formation of the cleavable complex is not completely reversed by EDTA when VM-26 is present, formation of double-strand breaks is nevertheless greatly reduced. Hence if topoisomerase II is responsible for the double-strand scissions in VM-26-treated KC cells, we should be able to suppress the formation of these cleavage products with EDTA. As described above, KC cells incubated with increasing amounts of VM-26 were harvested and split into two equal portions. One portion was lysed directly with SDS (see above), and the other was first treated with EDTA in the presence of the detergent Nonidet P-40 (NP40), which disrupts the cell membrane but does not induce DNA cleavage by topoisomerase II. As is evident from a comparison of the parallel SDS- and EDTA-terminated incubations in Fig. 1, EDTA substantially reduces the VM26-stimulated DNA cleavage reaction. At low levels of VM-26, most of the cleavage products observed in the SDS-lysed cells are absent or only weakly labeled when cells are preincubated with EDTA-NP-40 (Fig. 1, compare SDS and EDTA lanes 2 and 3). At higher levels of the drug (EDTA, lanes 5 and 6), some of the characteristic VM-26 fragments are observed; however, they are present at significantly lower levels than in the equivalent samples lysed directly with SDS (SDS, lanes 5 and 6). (ii) Suppression of the cleavage reaction by novobiocin. The antibiotic novobiocin interferes with the topoisomerase II catalytic cycle (8). It also reduces, but does not completely eliminate, DNA cleavage by the purified enzyme either in the absence (22) or in the presence (unpublished data) of VM-26. Hence it was of interest to determine whether novobiocin has any effect on the VM-26-stimulated DNA cleavage reaction in vivo in KC cells. To test this possibility, we divided KC cells into a series of equal portions. One set of cultures was pretreated for 15 min with 200 ,ug of novobiocin per ml and then incubated for an

TOPOISOMERASE II SITES OF ACTION

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FIG. 2. Novobiocin suppresses the VM-26-stimulated cleavage reaction. KC cells grown at 25°C were split into a series of equal portions. One group of cultures was pretreated with novobiocin at 200 ,ug/ml for 15 min and then incubated for 30 min with increasing concentrations of VM-26. The other set of cultures was incubated for 30 min with only VM-26. The cells were harvested and, after lysis with SDS, were treated with proteinase K in the presence of EDTA. The DNA was isolated, restricted with EcoRI and, after gel electrophoresis and blotting, probed with the EcoRI-SaII fragment from the proximal end of the 87A7 heat shock locus. Lanes with novobiocin (NOVO): 1, 100 ,ug of novobiocin per ml; 2, 75 ,ug of novobiocin per ml; 3, 50 ,ug of novobiocin per ml; 4, 25 p.g of novobiocin per ml. Lanes without novobiocin: 1, no VM-26; 2, 10 ,ug of VM-26 per ml; 3, 15 ,ug of VM-26 per ml; 4, 25 ,ug of VM-26 per ml; 5, 50 ,ug of VM-26 per ml; 6, 75 ,ug of VM-26 per ml; 7, 100 ,ug of VM-26 per ml; m, micrococcal nuclease. The asterisk between NOVO lanes 3 and 4 indicates (one or several) contaminating fragments which appear to be homologous to sequences (probably plasmid) in the probe. These fragments do not appear in many other blots with different DNA samples, and our experiments indicate that they are not induced by VM-26.

additional 30 min in the presence of increasing concentrations of VM-26. The other set of cultures was incubated only with VM-26. As can be seen from a comparison of the fragment patterns shown in Fig. 2, novobiocin suppresses the VM-26-stimulated cleavage reaction in tissue culture cells. DNA-protein linkage of the VM-26-induced cleavage products. DNA cleavage by topoisomerase II is accompanied by the formation of a covalent DNA-protein linkage at the 5' terminus of the cleavage site (17). If topoisomerase II is responsible for the double-strand scissions in drug-treated cells, an equivalent covalent DNA-protein linkage should be generated. A possible assay for such covalent complexes is suggested by previous studies on the purified enzyme (20).

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We found that the covalently linked topoisomerase II moiety markedly alters the electrophoretic migration of the cleavage products: the fragments containing the protein moiety give diffuse and irregular bands, and their mobility is retarded relative to that of the corresponding fragments in the proteinase K-treated control (20). In the experiments shown in Fig. 3, we used this gel assay to determine whether a similar stable DNA-protein complex is associated with the in vivo VM-26-induced cleavage products. For this purpose we first purified cellular DNA (and any DNA-protein complex) from SDS cell lysates by pelleting through either a glycerol-CsCl step gradient or a sucrose column. In Fig. 3A, DNA purified by using the glycerol-CsCl step gradient was restricted with EcoRI and then divided into two portions. One was deproteinized by digestion with proteinase K, and the other was not. As can be seen from inspection of the autoradiograph, all of the characteristic VM-26-induced cleavage products are observed in the aliquot treated with proteinase K prior to gel electrophoresis (lane 1). In addition, the mobility of these cleavage products is indistinguishable from that observed in DNA samples prepared by the standard isolation procedure (compare lanes 1 and 3). A quite different result is obtained for the alioquot electrophoresed directly without proteinase K treatment. Instead of a series of sharply defined bands, a much more diffuse smear of fragments is observed. Careful inspection of the autoradiograph indicates that that this smear consists of a series of discrete but broad and irregularly shaped bands. In each case, these bands appear to correspond to cleavage products found in the proteinase K-treated sample. Moreover, as was observed in experiments with purified topoisomerase II, the bands have a reduced electrophoretic mobility and are displaced upward relative to the corresponding cleavage products in the proteinase K-treated sample (see connecting lines). In other studies we have restricted the DNA samples with EcoRI plus restriction enzymes which cut within the 10-kb 87A7 EcoRI DNA segment and then analyzed the restriction fragments with and without proteinase K digestion. Unlike the VM-26induced cleavage products, the mobility of these restriction fragments is the same with or without proteinase K treatment (data not shown). In the experiment shown in Fig. 3B, DNA from control and VM-26-treated cells was isolated by pelleting through a sucrose column. In the DNA sample from untreated cells, there are no detectable cleavage products either with (lane 3) or without (lane 4) proteinase K digestion. In contrast, cleavage products are clearly evident in the DNA isolated from drug-treated cells. The fragment pattern is diffuse and upwardly displaced without proteinase K treatment, whereas the characteristic pattern of sharply defined bands is evident after proteinase K treatment. VM-26 induces a tight association of topoisomerase II with KC cell DNA. The experiments described in the previous section indicate that the SDS-mediated DNA cleavage reaction in VM-26-treated KC tissue culture cells is accompanied by the formation of a highly stable DNA-protein complex. If topoisomerase II is responsible for this cleavage reaction, the DNA-protein complexes observed in our experiments should consist of topoisomerase II molecules covalently linked to the DNA fragments. Hence, topoisomerase II would be expected to copurify with cellular DNA obtained from SDS lysates of KC cells treated with VM-26. In contrast, the enzyme would not be expected to copurify with cellular DNA from untreated cells. To test this possibility, KC DNA purified from SDS

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3 FIG. 3. VM-26 induces the formation of stable DNA-protein complexes. (A) KC cellular DNA was prepared by pelleting through a glycerol-CsCl step gradient as described in Materials and Methods. The pellet was resuspended in TE, and an aliquot was restricted with EcoRI. SDS was then added, and the sample was split into two parts. One part was digested with proteinase K (pr.k +), and the other was not (pr.k -). The samples were then loaded onto an agarose gel (in 0.5% SDS) and, after electrophoresis and blotting, probed with the EcoRI-SalI fragment. Lanes: M, micrococcal nuclease; 1, DNA prepared from VM-26-treated KC cells by the standard SDS lysis procedure; 2, DNA prepared from VM-26treated cells by pelleting through a glycerol-CsCl gradient, EcoRI restricted, and electrophoresed without proteinase K treatment; 3, DNA as in lane 2, except that the sample was treated with proteinase K after EcoRI restriction; 4, control DNA prepared from untreated KC cells restricted with EcoRI. (B) DNA was prepared from untreated (NO VM) and VM-26-treated (VM) KC cells by pelleting through a sucrose column as described in Materials and Methods. The DNA was restricted with EcoRI and split into two portions. One portion was digested with proteinase K (pr. k +), and the other was not (pr.k -). The samples were then loaded onto an agarose gel (in 0.5% SDS) and, after electrophoresis and blotting, probed with the proximal EcoRI-SalI fragment. Lanes: 1 and 2, EcoRl-restricted DNA purified from VM-26-treated cells undigested (-) or digested (+) with proteinase K; 3 and 4, EcoRI-restricted DNA purified from control cells which were not treated with VM-26; 5 and 6, another sample of DNA purified from VM-26-treated cells processed as in lanes 1 and 2. The asterisk indicates a contaminating band which hybridizes to the EcoRI-SalI probe (see legend to Fig. 2) and is observed in some DNA preparations. This band is not a VM-26-induced cleavage product (see no VM lanes), and its mobility is not greatly altered with or without proteinase K treatment.

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FIG. 4. Copurification of topoisomerase II with DNA isolated from VM-26-treated cells. KC cellular DNA prepared by centrifugation through a sucrose column as described in Materials and Methods and in the legend to Fig. 3B was digested with DNase I. The residual protein was then electrophoresed, blotted to nitrocellulose filters, and probed with rabbit anti-Drosophila topoisomerase II antibodies (a kind gift of M. Sander and T. Hsieh, Duke University). Lanes: 1, DNA purified from VM-26-treated KC cells; 2, DNA purified from control untreated cells; m, prestained molecular size markers.

lysates of untreated and VM-26-treated cells by pelleting through a sucrose column (see above) was digested with DNase I, and the residual protein was analyzed by Western immunoblotting with antibodies directed against purified Drosophila topoisomerase II. As can be seen in Fig. 4, lane 2, little or no cross-reacting material is detected in the

sample prepared from untreated KC cells. Thus, topoisomdoes not appear to cosediment with cellular DNA under our gradient centrifugation conditions. A different result is obtained for the protein sample prepared from VM-26-treated KC cells. In this case, the anti-Drosophila topoisomerase II serum stains a relatively prominent highmolecular-weight band (lane 1). The electrophoretic mobility of this band is consistent with a protein of molecular mass ca. 172 kDa. This is very close to the estimated monomeric molecular mass of purified Drosophila topoisomerase II, 170 kDa (17). In vivo versus in vitro cleavage products. In the autoradiographs shown in Fig. 5 we have compared the fragment pattern generated by purified topoisomerase II, using a naked 87A7 recombinant DNA substrate, with that generated by the endogenous topoisomerase II in isolated nuclei or in vivo in KC tissue culture cells. The results of this analysis for the DNA segment spanning the 5' ends of the hsp7O genes and the intergenic spacer region are summarized in the diagrams in Fig. 6. The positions of the major naked DNA sites for topoisomerase II in the intergenic spacer segment are indicated by the small boxes, and the positions of the major topoisomerase II sites in isolated nuclei (Topo in vitro) and in vivo in tissue culture cells (Topo in vivo) are indicated by the arrows. Also diagrammed in Fig. 6 is the chromatin structure of the intergenic spacer region deduced from the micrococcal nuclease cleavage pattern in isolated erase II

4977

nuclei (MN) and from studies with a variety of other nucleases (6, 21). It is clear from this analysis that the topoisomerase II cleavage pattern in naked DNA is very different from that observed either in nuclei or in vivo. Surprisingly, there are also some differences between the cleavage pattern in nuclei and in vivo. The salient findings are summarized below. (i) In naked DNA there are five major topoisomerase II cleavage sites in the intergenic spacer. However, these major naked DNA sites are not cleaved either in nuclei or in KC tissue culture cells (Fig. 6, Topo in vitro and Topo in vivo); instead, cleavage occurs at sites which are only secondary topoisomerase II sites in naked DNA. The lack of any cleavage products from the major naked DNA sites in nuclei and in tissue culture cells is presumably due to features of the nucleoprotein organization of the 87A7 locus, which limit the accessibility of these sites in chromatin. Consistent with this view, topoisomerase II cleavage in both nuclei and cells occurs at sites that are sensitive to micrococcal nuclease in chromatin digests (compare fragment patterns in Fig. SB and C). As indicated in Fig. 6, these cleavage sites correspond to the linker DNA sequences which separate adjacent nucleosomes in the intergenic spacer (21). In nuclei the yield of the fragments from the five internucleosomal linkers is nearly the same; however, this is clearly not the case for the in vivo topoisomerase II cleavage products. Instead, there is one very prominent band (band 5 in Fig. 5C) on the proximal side of the intergenic spacer, and two bands (bands 9 and 10) of moderate to low intensity on the distal side of the spacer. The remaining linkers are refractory to cleavage by topoisomerase II, and at most only very weakly labeled bands are observed. (ii) Although the 5' control region Znc contains naked DNA sites which are cleaved with only low or intermediate efficiency by purified topoisomerase II (Fig. SA), the major cleavage products in nuclei are derived from this DNA segment. As shown in Fig. SB and C, the first of the major nuclear cleavage products (band 3) is derived from the 5' end of the proximal transcription unit, whereas the second (band 4) is a closely spaced doublet derived from a sequence located ca. 170 to 190 bp upstream (Fig. SB and 6). Although poorly resolved in the indirect end-labeling experiment shown in Fig. 5, an equivalent pattern is observed for the distal Znc element (which is best visualized by using a SaII-BglII or a BglIH-EcoRI probe from the distal side of the locus [data not shown]). Although the same Znc fragments appear to be present in VM-26-treated KC cells, there are major differences in yield. Thus, cleavage at the doublet, site 4, is quite infrequent in tissue culture cells. In contrast, site 3, which spans the 5' end of the transcription unit, is quite sensitive in vivo and gives a relatively prominent band. Equivalent differences in the fragment pattern are observed for the distal Znc. (iii) In naked DNA the hsp7O transcription unit has only very minor topoisomerase II cleavage sites (Fig. SA). This is also true for nuclei prepared from cells grown at 25°C. As can be seen in Fig. SB, the hsp7O genes and the 3' proximal and distal spacer sequences are refractory to topoisomerase II cleavage, and only very minor bands are observed in these DNA segments even with high levels of VM-26 (Fig. SB) (20). A different result is obtained in vivo. First, two of the major topoisomerase II cleavage products in the heat shock locus are derived from the beginning portion of each hsp70 transcription unit. The cleavage sites for these fragments are spaced at nucleosome length intervals from the 5' cleavage site. The site (site 2) located closest to the 5' end is at ca.

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FIG. 5. Comparison of the topoisomerase II cleavage pattern in naked DNA, in isolated nuclei, and in vivo. (A) Topoisomerase II cleavage pattern in naked 87A7 DNA. Shown in this panel is the cleavage pattern generated by purified Drosophila topoisomerase II by using a naked substrate, the 10-kb 87A7 EcoRI fragment from the recombinant plasmid 122. The insert of plasmid 122 contains the two 87A7 hsp7O genes plus the flanking spacers and is equivalent to the 87A7 EcoRl fragment from KC tissue culture cells. In this experiment, EcoRI-linerarized plasmid was incubated either without (lane C) or with topoisomerase II (600 ng to 1.2 ,ug) and then processed as described in reference 22. The blot was probed with the proximal 87A7 EcoRI-SalI fragment. As a reference, 122 DNA was also partially digested with micrococcal nuclease (lane M) or SalI (lane S). (B) Topoisomerase II cleavage pattern in the 87A7 heat shock locus in isolated nuclei and in vivo. Shown in this panel is the topoisomerase II cleavage pattern induced in the 87A7 heat shock locus by VM-26 treatment of isolated nuclei and KC tissue culture cells. For the lanes labeled nuclei, DNA cleavage by the endogenous topoisomerase II in isolated nuclei was performed as described previously (23). KC cell nuclei were isolated and incubated with VM-26 (50 and 100 ,ug/ml) in the presence of ATP and MgCI2. The reaction was terminated by the addition of SDS, and the DNA was isolated by proteinase K digestion and phenol extraction. The DNA was restricted with EcoRI and, after electrophoresis and blotting, probed with the proximal EcoRI-SalI fragment. For the lanes labeled cell, in vivo topoisomerase II cleavage was as described in the legend to Fig. 1. For the lane labeled m, the micrococal nuclease cleavage pattern in chromatin is shown. DNA prepared from a micrococcal nuclease digestion of isolated KC cell nuclei was restricted with EcoRI. (C) Close-up of the 5' intergenic spacer region shown in panel B. The numbers are described in the text and in Fig. 6.

+220 bp, whereas the other site (site 1) is at ca. +440 bp (Fig. SB and 6). Although both of these fragments can also be detected in VM-26-treated nuclei, cleavage is much less frequent and only very weakly labeled bands are observed. Finally, there are some prominent naked DNA cleavage sites at the 3' boundaries of the hsp7O genes (Fig. 5A). By contrast, cleavage in this DNA segment both in isolated nuclei and in vivo is quite inefficient, and there are only some very minor fragments from this region. Localization of topoisomerase II around other genes. It was of interest to obtain a more complete picture of the distribution of topoisomerase II in vivo. For this purpose we selected two chromosomal DNA segments whose chromatin organization has been characterized in some detail. One of these is the Drosophila histone repeat unit (1, 16, 24), and the other is a tRNA gene cluster from the cytogenetic locus 9OBC (3).

(i) Histone repeat unit. The sequence organization of the histone repeat is shown in the diagram in Fig. 7. In previous studies with a recombinant containing the histone repeat unit (20), we found that there are many major naked DNA cleavage sites for topoisomerase II in the long H1-H3 spacer and at the 5' ends of each histone gene, whereas the transcribed regions have only very minor cleavage sites. Analysis of the chromatin structure of the repeat unit reveals that the long H1-H3 spacer which contains the most of the naked DNA topoisomerase II sites is assembled into an ordered nucleosomal array in which the core particles are aligned with respect to the underlying DNA sequence (1, 16, 24). Positioned nucleosomes are also found in the short H4-H2A and H2B-H1 spacers. The 5' ends of the five histones genes are hypersensitive to nuclease attack, and there appears to be an irregular distribution of nucleosomes within the genes. In the indirect end-labeling experiment

VOL. 11, 1991

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FIG. 6. Chromatin organization of the 87A7 intergenic spacer region and localization of the major topoisomerase II cleavage sites in naked DNA, isolated KC cell nuclei, and in vivo in KC tissue culture cells. (MN) This diagram shows the chromatin structure at the 5' ends of the two hsp7O genes and intergenic spacer region as deduced from an analysis of the nuclease digestion pattern in isolated nuclei (6, 21). Ovals indicate the approximate positions of nucleosomes. Arrows indicate the cleavage sites for micrococcal nuclease in chromatin. The dark arrows (e.g., arrow 3) correspond to the most prominent fragments, whereas the striped arrows (e.g., arrow 5) are somewhat less prominent. The small boxes in the intergenic spacer region (e.g., within nucleosome 5-6) correspond to the naked DNA cleavage sites for topoisomerase II. The SalI restriction sites mark the 5' end of each hsp7O transcription unit, and in each case the transcription unit extends toward the Cla and Bgl restriction sites. For each gene there is a nucleosome-free gap in chromatin spanning the region between approximately the Sall and Xba-Xho restriction sites (Znc) (Topo in vitro) This diagram shows the location of cleavage sites for endogenous topoisomerase II in isolated KC cell nuclei. The most prominent cleavage products are indicated by arrows 3 and 13 (dark arrows). The somewhat less prominent cleavage products (e.g., arrow 5) are indicated by striped arrows, while arrows 4 and 12 are both doublets. Other symbols are as described for MN above. (Topo in vivo) This diagram shows the location of the in vivo cleavage sites for topoisomerase II in KC tissue culture cells. The dark arrow (arrow 5) indicates the most prominent cleavage product. The stippled large arrows (e.g., arrow 9) indicate the positions of the moderate cleavage products, and the small arrows (arrows 4 and 6) indicate the position of cleavage products present in only very low yield. Other symbols are as described for MN above.

whose results are shown in Fig. 7, cells treated with increasing amounts of VM-26 were either lysed directly with SDS or treated with EDTA-NP-40 prior to the addition of SDS. The isolated cellular DNA was restricted with BgIIH and subsequently probed with a BglII-BamHI restriction fragment derived from within the Hi gene probe. As indicated, this displays the cleavage products reading from the Hi gene through the H1-H3 spacer toward the H3 and H4 genes. Despite the presence of some very strong topoisomerase II cleavage sites in the histone repeat in naked DNA, there appears to be little or no cleavage in the repeat unit in vivo, even at the highest concentration of VM-26. (By way of contrast, many more major as well as minor cleavage products are clearly evident in the heat shock locus at equivalent concentrations of the drug [compare Fig. 7 with Fig. 1]). In fact, the fragment pattern in samples from the SDS-lysed cells is nearly indistinguishable from that observed in control samples from cells pretreated with EDTA. The one possible

exception is a cleavage product derived from a linker separating adjacent nucleosomes in the H4-H2A spacer (16). This fragment is evident in the VM-26-treated cells lysed with SDS, but it is not present in the control EDTA-treated samples. In addition, there are two very minor fragments derived from two internucleosomal linkers in the long H1-H3 spacer (asterisks). (ii) tRNA gene cluster. One edge of the 9OBC tRNA gene cluster is shown in Fig. 8. As indicated in the diagram, this DNA segment has five tRNA genes arranged in two groups. In chromatin, each tRNA gene has a micrococcal and Si nuclease-hypersensitive region in the internal promoter (Fig. 8) (3). At the end of the cluster, there is a set of unusual micrococcal nuclease (and DNase I)-hypersensitive sites. These hypersensitive sites do not appear to be associated with a transcription unit (3), and the nuclease cleavage pattern resembles that found at the chromatin structures scs and scs', which flank the 87A7 heat shock locus (21). Unlike

4980

MOL. CELL. BIOL.

UDVARDY AND SCHEDL SDS

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FIG. 7. Topoisomerase II cleavage in the histone repeat unit. DNA was prepared from KC tissue culture cells treated with increasing amounts of VM-26, and the incubation was stopped with either SDS or EDTA as described in the legend to Fig. 1. The isolated DNA was restricted with BglII and, after electrophoresis and blotting, probed with a BglII-BamHI fragment from inside the histone Hi gene (16). As indicated in the diagram, this displays the cleavage products reading from the Hi gene through the long H1-H3 spacer into H3-H4 and H2A-H2B. Lanes: 1, no VM-26; 2, 10 ,ug of VM-26 per ml; 3, 25 ,ug of VM-26 per ml; 4, 50 ,ug of VM-26 per ml; 5, 100 ,ug of VM-26 per ml; M, DNA prepared from a micrococcal nuclease digest of isolated KC cell nuclei and restricted with BgIII. The organization of nucleosomes in the histone repeat deduced from micrococcal nuclease and DNase I digestion is reported in references 16 and 25. Asterisks indicate the positions of VM-26-induced cleavage products (see the text).

the histone repeat, the 9OBC tRNA locus has some relatively major in vivo sites for topoisomerase II cleavage. The topoisomerase II cleavage products can be identified by comparing the DNA fragments obtained after cell lysis with SDS or EDTA-NP-40. The major topoisomerase II-induced cleavage products are derived from the region upstream of tRNAVaI, and map within or close to the chromatin-specific micrococcal nuclease- and DNase I- hypersensitive sites (3) located in this DNA segment. In addition, topoisomerase II cleaves at two minor sites located between (or 3' to) the tRNAVa' and tRNAPrO genes. In contrast, there are no topoisomerase II cleavage sites associated with the group of three downstream tRNA genes; although weakly labeled fragments are observed within the internal promoters of each of these tRNA genes, the relative yield of the fragments is

FIG. 8. Topoisomerase II cleavage in the 9OBC tRNA gene cluster. DNA was prepared from KC tissue culture cells treated with increasing amounts of VM-26, and the incubation was stopped with either SDS or EDTA as described in the legend to Fig. 1. The DNA was restricted with BamHI and, after electrophoresis and blotting, probed with a BamHI-XhoI fragment (probe K) from 9OBC. As described previously (3), this displays the cleavage products reading first through a cluster of three tRNA genes (Ala, Pro, and Ala) and then through a second cluster of two tRNA genes (Val and Pro). Lanes: 1, no VM-26; 2, 5 ,ug of VM-26 per ml; 3, 10 ,ug of VM-26 per ml; 4, 25 pug of VM-26 per ml; 5, 50 ,ug of VM-26 per ml; 6, 100 ,ug of VM-26 per ml; M, DNA prepared from a micrococcal nuclease digest of isolated KC cell nuclei; Si, DNA from an Si nuclease digestion of isolated KC cell nuclei. EDTA, EDTA-NP-40-terminated incubation. SDS, SDS-terminated incubation. Both Si and micrococcal nuclease have prominent chromatin-specific cleavage sites in the internal control region of the five 9OBC tRNA genes shown here. not drug dependent (compare lanes 1 and 6) is not obviously reduced when cells are lysed with EDTA.

DISCUSSION Proteins which interact with specific DNA sequences play critical role in regulatory processes in both eukaryotes and prokaryotes. Over the past decade a large number of different eukaryotic DNA-binding proteins have been identified and characterized (see, e.g., reference 9). Many of these a

VOL. 1 l, 1991

regulatory proteins are members of gene families which have common structural motifs like the helix-turn-helix of the homeo-domain. Moreover, these proteins often recognize quite similar DNA sequences. For example, several of the Drosophila homeo-domain proteins interact in vitro with the DNA sequence TCAATTAAAT or variants of this sequence (4, 7). Surprisingly, the homeo-domain proteins (as well as proteins from some of the other gene families) also appear to tolerate a considerable degree of DNA sequence variation and are able to recognize sites in vitro whose sequence differs significantly from the consensus. The rather low degree of sequence specificity observed in vitro poses a difficult question: how can such apparently indiscriminate proteins function in specific regulatory circuits in vivo? As an example, sequences resembling the homeo-domain recognition site would be expected to occur quite frequently in stretches of A+T rich DNA such as the spacer DNA segments at the 87A7 heat shock locus or in the histone repeat. Indeed, there are at least six sites in the 87A7 intergenic spacer which closely conform to the above homeo-domain consensus and hence might be expected to interact with homeo-domain proteins in vitro. On the other hand, there is little reason to believe that any of the homeo-domain-binding sites at the heat shock locus are functional. Hence, to understand how specificity is achieved in eukaryotic regulatory systems, it will be important to determine which, if any, of the in vitro-defined sites for the regulatory proteins can be occupied and/or are functionally relevant in vivo. This problem is not easily addressed for many eukaryotic regulatory proteins; however, it is possible to compare in vitro and in vivo sites for the enzyme topoisomerase II. Even though topoisomerase II is considerably more promiscuous in its interactions with DNA than a typical homeodomain-type regulatory protein, the Drosophila enzyme has a surprisingly high degree of sequence specificity. This first became evident in the analysis of sites utilized by purified topoisomerase II in the SDS-mediated DNA cleavage reaction (10, 17, 18, 20). For example, some DNA segments, such as the 87A7 intergenic spacer, have clusters of very strong cleavage sites, whereas other segments, such as the 87A7 hsp7O genes, have only weak sites. Subsequent experiments have shown that the enzyme displays the same specificity in its selection of DNA-binding sites and sites for the catalysis of strand passage events during the relaxation of supercoiled molecules (17). The sequence specificity deduced from mapping the DNA cleavage sites has been confirmed by DNase I footprinting (10). The enzyme protects a DNA segment of about 23 bp, and the footprints cover sequences corresponding to the strong topoisomerase II DNA cleavage sites (10). Hence, not unlike a "typical" eukaryotic regulatory protein, the intrinsic sequence specificity of topoisomerase II largely determines how it interacts with DNA substrates in vitro. It would not be unreasonable to suppose that this intrinsic sequence specificity will also be of considerable importance in the functioning of topoisomerase II in vivo in processes which require alterations in (or the maintenance of) the topological state of the DNAchromatin fiber. In the experiments presented here we have determined the sites of action of topoisomerase II in vivo in several different chromosomal DNA segments by mapping DNA cleavage sites. For this purpose we used the topoisomerase II inhibitor VM-26 to stimulate the formation of double-strand scissions in Drosophila KC tissue culture cells. Several lines

TOPOISOMERASE II SITES OF ACTION

4981

of evidence would suggest that topoisomerase II is responsible for generating these double-strand scissions. (i) Purified topoisomerase II generates strand scissions (in the presence or absence of VM-26) when enzyme-DNA complexes are denatured by SDS (or other reagents), whereas DNA cleavage is substantially suppressed when the complexes are pretreated with EDTA prior to the addition of SDS. This is also the case for the VM-26-induced cleavage products in KC cells: we have found that double-strand breaks are observed in genomic DNA isolated from VM-26treated KC tissue culture cells when the cells are lysed with SDS; in contrast, preincubation of the VM-26-treated cells with the chelator EDTA (in the presence of the detergent NP-40 to disrupt the cell membranes) suppresses the formation of double-strand scissions. (ii) In experiments with purified topoisomerase II, the antibiotic novobiocin was found to partially inhibit the DNA cleavage reaction. Novobiocin also reduces the extent of DNA cleavage in VM-26-treated KC cells. (iii) The SDS-mediated cleavage of DNA by topoisomerase II is accompanied by the covalent attachment of a denatured protein moiety to the DNA. The formation of this covalent protein-DNA complex can be monitored by comparing the electrophoretic mobility of the topoisomerase II cleavage products with and without proteinase K digestion. We have used this assay to analyze the cleavage products from VM-26-treated KC cells. Our experiments indicate that the VM-26-induced cleavage products from tissue culture cells are also associated with a very stable (and most probably covalent) protein-DNA complex. (iv) Finally, we have found that topoisomerase II copurifies with DNA isolated from SDS lysates of VM-26-treated KC cells. In contrast, the enzyme does not copurify with genomic DNA from untreated control cells. These findings, together with the results of our previous studies on VM-26induced DNA cleavage in isolated nuclei (22), would implicate topoisomerase II in the formation of double-strand breaks in VM-26-treated KC tissue culture cells. Topoisomerase II sites of action are restricted in vivo. A comparison of the cleavage pattern generated in vitro by purified topoisomerase II on naked DNA substrates with that obtained in vivo indicates that the potential sites of action for topoisomerase II are very much restricted inside the cell. Moreover, the primary targets for the enzyme in naked DNA are of little value in predicting which sites are likely to be targets for the enzyme in vivo. In fact, in the DNA segments we have examined, very few, if any, of the strong naked DNA sites appear to be cleaved in vivo; instead, cleavage occurs at secondary naked DNA sites. The discrepancy between the naked DNA and in vivo cleavage patterns cannot be explained simply by an inability of VM-26 to stimulate cleavage at strong naked DNA sites. In the 87A7 intergenic spacer, for example, the drug clearly enhances cleavage by the purified enzyme at the strong naked DNA sites, and drug stimulation of the cleavage reaction is first evident at these sites. However, the same strong naked DNA sites are not cleaved by the enzyme in the presence of VM-26 in vivo or even in isolated nuclei. The differences between the naked DNA and in vivo cleavage patterns also cannot be explained by effects of the drug on the fidelity of the cleavage reaction. Although some minor alterations in the relative cleavage efficiency at specific sites are evident on DNA sequencing gels, VM-26 does not grossly perturb the specificity of the cleavage reaction. Moreover, these variations are not readily detected on agarose gels, and in our experiments the selection of cleav-

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UDVARDY AND SCHEDL

age sites by the purified enzyme on naked DNA substrates appears to be essentially the same with or without the drug

(22). A more plausible explanation for why the in vivo cleavage pattern is different from that in naked DNA comes from our previous studies on the chromatin structure of the 87A7 heat shock locus (21), the histone repeat unit (1, 16, 24), and the 9OBC tRNA gene cluster (3). We found that each of these chromosomal segments is organized into a defined and highly ordered nucleoprotein structure. This nucleoprotein organization limits access to the DNA, and cleavage by a variety of nucleases is restricted to hypersensitive regions associated with promoter elements (5' or internal) and to the linker DNA segments separating adjacent nucleosomes. The 87A7 intergenic spacer, for example, appears to be packaged into an ordered nucleosome array in which each core particle occupies a more or less well-defined position along the DNA (21) (Fig. 6). A comparison of the map positions of these nucleosome core particles with the location of the strong topoisomerase II naked DNA cleavage sites reveals that in virtually all cases the strong cleavage sites are located in DNA sequences which appear to be wrapped around the nucleosome core particle (Fig. 6, boxes). This is also true for the long H1-H3 spacer of the histone repeat unit. This spacer contains most of the strong naked DNA topoisomerase II cleavage sites (20), and from our previous chromatin studies it appears that many of these sites are also located in nucleosome core DNA sequences (16). It would not be unreasonable to suppose that the association of a strong cleavage site with the core particle may effectively preclude any productive interactions with topoisomerase II. The path of DNA wrapped around the surface of the nucleosome core particle selectively exposes or protects nucleotide residues in major and minor grooves with a periodicity roughly that of B-form DNA, 10.4 bp. Since topoisomerase II is a rather large enzyme and protects more than two turns of the B-form helix, essential contacts for the enzyme along the DNA duplex may be inaccessible either because they are oriented toward the surface of the nucleosme or because of steric hinderence. In contrast to the DNA wrapped around the nucleosome, DNA in the linker regions separating adjacent core particles is typically much more sensitive to nuclease attack. Consequently, the linker regions could be in a configuration in which the major and/or minor grooves are readily accessible over one or more turns of the helix. In this case, these sequences would potentially be available for productive interactions with proteins such as topoisomerase II. Consistent with this possibility, all of the in vivo cleavage products in the 87A7 intergenic spacer map to internucleosomal linkers. As expected from the intrinsic sequence specificity of the enzyme, cleavage in the linkers occurs at sites which are also used in naked DNA; however, in the 87A7 intergenic spacer, these linker sites are only minor naked DNA sites, not the very strong naked DNA sites. Topoisomerase II does not, however, cleave efficiently in all linker sequences in vivo. As indicated in the diagram in Fig. 6, only very weakly labeled cleavage products are derived from linkers 6/7 and 8 in the 87A7 intergenic spacer. The linkers separating aligned nucleosome core particles in the long H1-H3 histone repeat spacer are even more refractory to topoisomerase II cleavage in VM-26-treated cells, and at most, only very minor fragments are evident in this DNA segment. For the 87A7 intergenic spacer, the lack of fragments from linkers 6/ and 8 cannot be due to an inability of VM-26 to stimulate cleavage at sites in these DNA

MOL. CELL. BIOL.

segments. As indicated in Fig. 6, these linkers are cleaved with reasonable efficiency in VM-26-treated nuclei. The differences in linker sensitivity are probably related to the length of the nucleosomal unit. In our analysis of the chromatin organization of the 87A7 intergenic spacer (21), we found several cases in which the linker-to-linker interval (as determined with micrococcal nuclease) appeared to be somewhat larger than the "typical" 180- to 185-bp Drosophila nucleosomal unit. The nucleosomal units defined by linkers 4-5 and 10-11/12 are more than 250 bp in length, whereas the 5-6/7 unit is 230 bp and the 9-10 unit is 200 bp. As can be seen in the diagram in Fig. 6, the strong topoisomerase II cleavage products in the intergenic spacer map to the linkers of these larger nucleosomal units. In contrast, the nucleosomal units defined by linkers 6/7-8 and 8-9 are smaller than average (only about 160 to 170 bp) and topoisomerase II cleavage at these linkers is quite inefficient in vivo. The lengths of the nucleosomal units may also explain why the H1-H3 spacer of the histone repeat has little topoisomerase II cleavage in vivo (and in isolated nuclei). Unlike the 87A7 intergenic spacer, the histone spacer contains no extraordinarily large nucleosomal units, and the linker-to-linker intervals of the six units in the spacer range from 180 to 210 bp (1, 16, 24). Of these, only the 210-bp unit shows any topoisomerase II cleavage at the surrounding linkers in vivo (asterisks in Fig. 6), and this cleavage is not much above background. The 87A7 heat shock locus has several other major in vivo topoisomerase II cleavage sites in addition to those in the intergenic spacer. There are three major sites around the 5' end of each hsp7O transcription unit which are located in sequences that have only very weak cleavage sites in naked DNA. The first of these sites maps at the downstream edge of the 5' hypersensitive region. Topoisomerase II sites have also mapped to the 5' nuclease-sensitive regions in the chicken ,B-globin locus (13, 14). The second and third sites are within the gene and map ca 220 and ca 440 bp, respectively, from the 5' nucleosome-free gap. Both of these sites are located in linker segments separating aligned nucleosomes (21). This set of three cleavage sites may be a common feature of the Drosophila hsp7O genes. Using a probe which visualizes the 5' region of all Drosophila hsp7O genes (a fragment derived from within the gene), Rowe et al. (15) detected three topoisomerase II cleavage products which closely resemble those that we have observed for the proximal and distal 87A7 genes. Quite different results are obtained for the 5' hypersensitive regions of the histone genes. Even though the 5' ends of all five histone genes have prominent naked DNA sites for topoisomerase 11 (20), little or no cleavage is evident at these sites either in vivo or in isolated nuclei (22). In contrast, micrococcal nuclease, which appears to recognize the same 5' sites in naked DNA as topoisomerase 11 (20), is able to cut at these sites in chromatin (16). Since micrococcal nuclease is a much smaller enzyme, it conceivable that some type of steric hinderance, either from nearby nucleosomes or from various regulatory proteins, prevents topoisomerase II from interacting with the histone 5' sites. In the latter case it may be of interest that the internal promoter elements of the tRNA genes at 9OBC are also refractory to topoisomerase II cleavage in vivo. The formation of very stable transcription complexes on the internal control elements of a variety of RNA polymerase III genes is well documented. Such complexes might preclude interaction with topoisomerase II while permitting cleavage by much smaller enzymes such as

TOPOISOMERASE II SITES OF ACTION

VOL. 1 l, 1991

micrococcal nuclease, S1, or DNase I (3). Similar findings have been reported for the P2-tubulin promoter (14). The in vivo pattern differs from that in isolated nuclei. One unexpected observation is that the yield of cleavage products from specific sites in the 87A7 heat shock in vivo is different from that obtained in isolated nuclei. For example, in vivo topoisomerase II cleaves preferentially at only a subset of the internucleosomal linkers in the intergenic spacer. In contrast, in nuclei all of these linkers are cleaved with nearly the same efficiency. Differences are also apparent at the 5' end of the hsp genes. In vivo there are two major sites, one at ca. +220 bp and the other at ca. +440 bp. In contrast, in nuclei there is little cleavage within the gene and only quite minor fragments are generated from these two sites. The differences between the in vivo and nuclear cleavage patterns are likely to reflect a change in the relative accessibility of the cleavage sites. This would suggest that the native in vivo nucleoprotein organization of the heat shock locus may be partially disrupted by the procedures used to isolate nuclei. In the intergenic spacer, for example, some of the nucleosomal units are unusually large, and it is conceivable that the chromatin architecture of this DNA segment may be especially susceptible to mechanical or ionic perturbations. The more uniform nuclear pattern of topoisomerase II cleavage in the spacer would then arise either because the three-dimensional organization of these nucleosomes is altered or because the nucleosomes slide during the preparation and/or incubation of the nuclei. Changes in the chromatin architecture might also be facilitated by the loss of nonhistone proteins. Similar mechanisms might explain the differences in yield of cleavage products from Znc and from the first part of the hsp7O transcription unit. For example, the inefficient cleavage at the two sites inside the transcription unit (ca. +220 and ca. +440 bp) in nuclei might be explained by a rearrangement of the nearby nucleosome core particles. It is also possible that some nonhistone protein(s) is required to facilitate the binding of topoisomerase II at these two sites in vivo. In this case, such a protein might dissociate from the DNA in isolated nuclei. A change in relative accessibility alone would not, however, completely account for the differences in cleavage pattern. In addition, we must suppose (i) that at least some fraction of the bound topoisomerase II dissociates from the DNA (either during the isolation of nuclei or during the incubation with VM-26) and (ii) that free topoisomerase II molecules in the nuclei interact with newly accessible binding sites. These suppositions would be consistent with previous studies on purified topoisomerase II, which indicated that enzyme-DNA complexes have a finite half-life (several minutes), which depends upon both the ionic conditions and the affinity of the binding sites (17). It should be pointed out that in view of the rather substantial differences between the topoisomerase II cleavage sites in vivo, in nuclei, and in naked DNA, considerable caution should be exercised in interpreting the results of in vitro binding protocols which have implicated topoisomerase II in matrix attachment. In these experiments, most of the chromosomal proteins are stripped off from the DNA by high salt or other sorts of treatment (5). If topoisomerase II remains active under such conditions, it would be expected to dissociate from many of the low-affinity binding sites that constitute the actual targets of the enzyme in vivo. It would then preferentially interact with the newly exposed highaffinity naked DNA sites such as those found in the 87A7

4983

intergenic spacer or the H1-H3 spacer of the histone repeat (19, 22). Chromatin structure and in vivo specificity. The results presented here indicate that the interaction of topoisomerase II with specific sequences in vivo is dictated by the chromatin structure of the DNA segment. All of the in vivo cleavage sites appear to be restricted to DNA sequences which are in an accessible configuration, either in nuclease-hypersensitive regions or in longer-than-average internucleosomal linkers. In contrast, sequences associated with the nucleosome particle, as well as sequences in most linkers between nucleosomes, are apparently unavailable in vivo. The con-

straints imposed in vivo on topoisomerase II sites of action raise the more general question of whether chromatin structure also dictates how other proteins interact with DNA in vivo. It is true that topoisomerase II is a rather large molecule and may be especially sensitive to restrictions in DNA accessibility and conformation which arise when the DNA is organized into nucleosomes. On the other hand, it seems likely that the factors which limit sites of action for topoisomerase II in chromatin will also serve to constrain many other protein-DNA interactions in vivo. From this perspective, nucleosome organization (be it positioned or nonpositioned nucleosomes) may provide a very important and widely used mechanism for generating specificity in the interactions of regulatory proteins with potential target sequences. ACKNOWLEDGMENTS This work was supported by NIH grants to P.S. P.S. also thanks the March of Dimes for supporting part of this work. The drug VM-26 was a kind gift of Bristol Myers-Squibb. We especially thank M. Sanders and T. Hsieh, Duke University, for the

gift of Drosophila topoisomerase II and of anti-topoisomerase II antibody. We also thank S. Han and K. Udvardy for excellent technical assistance. REFERENCES 1. Cartwright, I., R. P. Hertzberg, P. B. Dervan, and S. C. R. Elgin. 1983. Cleavage of chromatin with methidiumpropylEDTA iron (II). Proc. Natl. Acad. Sci. USA 80:3213-3217. 2. Chen, G. L., L. Yang, T. C. Rowe, B. D. Halligan, K. M. Tewey, and L. F. Liu. 1984. Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J. Biol. Chem. 259:13560-13566. 3. DeLotto, R., and P. Schedl. 1984. The internal promotor elements of tRNA genes are preferentially exposed in chromatin. J. Mol. Biol. 179:607-628. 4. Desplan, C., J. Thesis, and P. H. O'Farrell. 1988. The sequence specificity of homeodomain-DNA interactions. Cell 54:1081-

1090. 5. Gross, D. S., and W. T. Garrard. 1988. Nuclease hypersensitive sites in chromatin. Annu. Rev. Biochem. 57:159-197. 6. Han, S., A. Udvardy, and P. Schedi. 1984. Transcriptionally active chromatin is sensitive to Neurospora crassa and S1

nucleases. J. Mol. Biol. 179:469-496. 7. Hoey, T., and M. Levine. 1988. Divergent homeo box proteins recognize similar DNA sequences in Drosophila. Nature (London) 332:858-861. 8. Hsieh, T., and D. Brutlag. 1980. ATP dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings. Cell 21:115-125. 9. Johnson, P. F., and S. L. McKnight. 1989. Eukaryotic transcriptional regulatory proteins. Annu. Rev. Biochem. 58:799-839. 10. Lee, M. P., M. Sander, and T. Hsieh. 1989. Nuclease protection by Drosophila DNA topoisomerase II. J. Biol. Chem. 264:

21779-21787. 11. Liu, L. F. 1983. DNA topoisomerases-enzymes that catalyze the breaking and rejoining of DNA. Crit. Rev. Biochem. 15:124.

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12. Liu, L. F. 1989. DNA topoisomerase poisons as antitumor drugs. Annu. Rev. Biochem. 58:351-375. 13. Muller, M. T., and V. B. Mehta. 1988. DNase I hypersensitivity is independent of endogenous topoisomerase II activity during chicken erythrocyte differentiation. Mol. Cell. Biol. 8:36613669. 14. Reitman, M., and G. Felsenfeld. 1990. Developmental regulation of topoisomerase II sites and DNase I hypersensitive sites in the chicken ,-globin locus. Mol. Cell. Biol. 10:2774-2786. 15. Rowe, T. C., J. C. Wang, and L. F. Liu. 1986. In vivo localization of DNA topoisomerase II cleavage sites on Drosophila heat shock chromatin. Mol. Cell. Biol. 6:985-992. 16. Samal, B., A. Worcel, C. Louis, and P. Schedl. 1982. Chromatin structure of the histone genes of Drosophila melanogaster Cell 23:401-409. 17. Sander, M., and T. Hsieh. 1983. Double strand cleavage by type II DNA topoisomerase purified from Drosophila melanogaster. J. Biol. Chem. 258:8421-8428. 18. Sander, M., and T. Hsieh. 1985. Drosophila topoisomerase II double-strand cleavage: analysis of DNA sequence homology at the cleavage site. Nucleic Acids Res. 13:1057-1072.

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19. Sander, M., T. Hsieh, A. Udvardy, and P. Schedl. 1987. Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding. J. Mol. Biol. 194:219-229. 20. Udvardy, A., E. Maine, and P. Schedl. 1985. The 87A7 chromomere: identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains. J. Mol. Biol. 185:341-358. 21. Udvardy, A., and P. Schedl. 1984. Chromatin organization of the 87A7 heat shock locus of Drosophila melanogaster. J. Mol. Biol. 172:385-403. 22. Udvardy, A., P. Schedl, M. Sander, and T. Hsieh. 1985. Novel partitioning of DNA cleavage sites for Drosophila topoisomerase II. Cell 40:933-941. 23. Udvardy, A., P. Schedl, M. Sander, and T. Hsieh. 1986. Topoisomerase II cleavage in chromatin. J. Mol. Biol. 191:231-246. 24. Wang, J. 1985. DNA topoisomerases. Annu. Rev. Biochem. 54:665-697. 25. Worcel, A., G. Gargiulo, B. Jessee, A. Udvardy, C. Louis, and P. Schedl. 1983. Chromatin fine structure of the histone gene complex of Drosophila melanogaster. Nucleic Acids Res. 11: 421-439.