Drug Stimulated DNA Cleavage Mediated by Cauliflower Topoisomerase II

We have investigated cauliflower (Brassica oleracea) topoisomerase II with respect to its interaction with DNA and demonstrate that the enzyme shares the characteristics of topoisomerase II purified from a variety of phylogenetically remote organisms. In the presence of the 2-nitroimidazole Ro 15-0216, cauliflower topoisomerase II-mediated DNA cleavage is extensively stimulated (approximately 20-fold) only at a site recognized as a major cleavage site for the enzyme in the absence of drug. The conservation of the enzyme's DNA specificity in the presence of Ro 15-0216 is in contrast to the effect exerted by traditional topoisomerase II inhibitors, which cause enzyme-mediated cleavage to take place at a multiple number of DNA sites. Ro 15-0216 may therefore prove useful as a tool in the elucidation of the enzyme's DNA interaction sites and its involvement in nucleic acid metabolism in plant cells.     

Mode of action of topoisomerase II-targeting agents at a specific DNA sequence. Uncoupling the DNA binding, cleavage and religation events.

Methods of uncoupling the DNA binding, cleavage and religation reactions of topoisomerase II were employed to investigate the influence of topoisomerase II-directed drugs on the individual steps in the enzyme's catalytic cycle. A special DNA substrate containing a major topoisomerase II interaction site, which can be cleaved by the enzyme in the absence of any concomitant religation, was used to examine the effect of topoisomerase II-directed agents upon the DNA cleavage reaction. The experiment demonstrated that the topoisomerase II targeting agent Ro 15-0216 stimulates the DNA cleavage reaction extensively, whereas the traditional topoisomerase II inhibitor, mAMSA, has only a minor effect on this reaction. Topoisomerase II trapped in the cleavage complexes can religate to the 3' hydroxyl end of another DNA strand. Using this religation assay, it was demonstrated that the major effect of mAMSA is an inhibition of the enzyme's religation reaction, whereas Ro 15-0216 has no effect on this reaction. Recently, considerable attention has been given to drugs preventing topoisomerase II from introducing DNA cleavages. In the present paper the initial non-covalent DNA binding reaction of topoisomerase II was investigated under conditions excluding enzyme-mediated DNA cleavage. This demonstrated that the anthracycline, aclarubicin, prevents topoisomerase II from performing its initial non-covalent DNA binding reaction and thereby abolishes the DNA cleavage reaction of the enzyme. The results presented here demonstrate that profound differences exist in the mode of action of different agents targeting topoisomerase II, and that the enzyme can be affected by such agents at both its DNA binding, cleavage and religation subreactions.     

Sequence-dependent effect of camptothecin on human topoisomerase I DNA cleavage

We have studied the effect of the antitumor drug, camptothecin, on the interaction of human topoisomerase I with DNA at the sequence level. At a low molar ratio of enzyme to DNA, cleavage is prominent and unique, located at a previously described hexadecameric recognition sequence, while a number of strong additional cleavage sites appear in the presence of the drug. Camptothecin stimulates cleavage at the recognition sequence less than twofold, whereas cleavage at the additional sites is stimulated up to 200-fold. Camptothecin greatly enhances the stability of the cleavable complexes formed at the additional sites, whereas the complex formed at the hexadecameric sequence is only marginally affected. Cleavage was eliminated at certain sites in the presence of camptothecin. Taken together these observations demonstrate that at least three types of potential eukaryotic topoisomerase I cleavage sites can be distinguished by the use of camptothecin. Comparison of the sequences at the additional cleavage sites in the presence of camptothecin reveals that the most frequently cleaved dinucleotide is TG with no consensus for the flanking nucleotides.     

Stabilization of the topoisomerase II-DNA cleavage complex by antineoplastic drugs: inhibition of enzyme-mediated DNA religation by 4'-(9-acridinylamino)methanesulfon-m-anisidide

In order to elucidate the mechanism by which the intercalative antineoplastic drug 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) stabilizes the covalent topoisomerase II-DNA cleavage complex, the effect of the drug on the DNA cleavage/religation reaction of the type II enzyme from Drosophila melanogaster was examined. At a concentration of 60 microM, m-AMSA enhanced topoisomerase II mediated double-stranded DNA breakage approximately 5-fold. Drug-induced stabilization of the enzyme-DNA cleavage complex was readily reversed by the addition of EDTA or salt. When a DNA religation assay was utilized, m-AMSA was found to inhibit the topoisomerase II mediated rejoining of cleaved DNA approximately 3.5-fold. This result is similar to that previously reported for the effects of etoposide on the activity of the Drosophila enzyme [Osheroff, N. (1989) Biochemistry 28, 6157-6160]. Thus, it appears that structurally disparate classes of topoisomerase II targeted antineoplastic drugs stabilize the enzyme's DNA cleavage complex primarily by interfering with the ability of topoisomerase II to religate DNA.     

1,4-Benzoquinone is a topoisomerase II poison

Benzene is a human carcinogen that induces hematopoietic malignancies. It is believed that benzene does not initiate leukemias directly, but rather generates DNA damage through a series of phenolic metabolites, especially 1,4-benzoquinone. The cellular consequences of 1,4-benzoquinone are consistent with those of topoisomerase II-targeted drugs. Therefore, it has been proposed that the compound initiates specific leukemias by acting as a topoisomerase II poison. This hypothesis, however, has not been supported by in vitro studies. While 1,4-benzoquinone has been shown to inhibit topoisomerase II catalysis, increases in enzyme-mediated DNA cleavage have not been reported. Because of the potential involvement of topoisomerase II in benzene-induced leukemias, we re-examined the effects of the compound on DNA cleavage mediated by human topoisomerase IIalpha. In contrast to previous reports, we found that 1,4-benzoquinone was a strong topoisomerase II poison and was more potent in vitro than the anticancer drug etoposide. DNA cleavage enhancement probably was unseen in previous studies due to the presence of reducing agents in reaction buffers and the incubation of 1,4-benzoquinone with the enzyme prior to the addition of DNA. 1,4-Benzoquinone increased topoisomerase II-mediated DNA cleavage primarily by enhancing the forward rate of scission. In vitro, the compound induced cleavage at DNA sites proximal to a defined leukemic chromosomal breakpoint and displayed a sequence specificity that differed from that of etoposide. Finally, 1,4-benzoquinone stimulated DNA cleavage by topoisomerase IIalpha in cultured human cells. The present findings are consistent with the hypothesis that topoisomerase IIalpha plays a role in the initiation of specific leukemias induced by benzene and its metabolites.     

Cobalt enhances DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells

Although cobalt is an essential trace element for humans, the metal is genotoxic and mutagenic at higher concentrations. Treatment of cells with cobalt generates DNA strand breaks and covalent protein-DNA complexes. However, the basis for these effects is not well understood. Since the toxic events induced by cobalt resemble those of topoisomerase II poisons, the effect of the metal on human topoisomerase IIalpha was examined. The level of enzyme-mediated DNA scission increased 6-13-fold when cobalt(II) replaced magnesium(II) in cleavage reactions. Cobalt(II) stimulated cleavage at all DNA sites observed in the presence of magnesium(II), and the enzyme cut DNA at several "cobalt-specific" sites. The increased level of DNA cleavage in the presence of cobalt(II) was partially due to a decrease in the rate of enzyme-mediated religation. Topoisomerase IIalpha retained many of its catalytic properties in reactions that included cobalt(II), including sensitivity to the anticancer drug etoposide and the ability to relax and decatenate DNA. Finally, cobalt(II) stimulated topoisomerase IIalpha-mediated DNA cleavage in the presence of magnesium(II) in purified systems and in human MCF-7 cells. These findings demonstrate that cobalt(II) is a topoisomerase II poison in vitro and in cultured cells and suggest that at least some of the genotoxic effects of the metal are mediated through topoisomerase IIalpha.     

Effect of antineoplastic agents on the DNA cleavage/religation reaction of eukaryotic topoisomerase II: inhibition of DNA religation by etoposide

Beyond its essential physiological functions, topoisomerase II is the primary cellular target for a number of clinically relevant antineoplastic drugs. Although the chemotherapeutic efficacies of these drugs correlate with their abilities to stabilize the covalent topoisomerase II-DNA cleavage complex, their molecular mechanism of action has yet to be described. In order to characterize the drug-induced stabilization of this enzyme-DNA complex, the effect of etoposide on the DNA cleavage/religation reaction of Drosophila melanogaster topoisomerase II was studied. Under the conditions employed, etoposide increased levels of enzyme-mediated double-stranded DNA cleavage 5-6-fold and single-stranded cleavage approximately 4-fold. Maximal stimulation was observed at 80-100 microM etoposide with 50% of the maximal effect at approximately 15 microM drug. By employing a topoisomerase II mediated DNA religation assay [Osheroff, N. & Zechiedrich, E.L. (1987) Biochemistry 26, 4303-4309], etoposide was found to stabilize the enzyme-DNA cleavage complex (at least in part) by inhibiting the enzyme's ability to religate cleaved DNA. Moreover, in order for the drug to affect religation, it has to be present at the time of DNA cleavage.     

Mammalian topoisomerase II activity is modulated by the DNA minor groove binder distamycin in simian virus 40 DNA

DNA topoisomerases II are nuclear enzymes that have been identified recently as targets for some of the most active anticancer drugs. Antitumor topoisomerase II inhibitors such as teniposide (VM-26) produce enzyme-induced DNA cleavage and inhibition of enzyme activity. By adding to such reactions distamycin, a compound whose effects on DNA have been extensively characterized, we investigated the effects of drug binding upon topoisomerase II-mediated DNA cleavage induced by VM-26. We have found a correspondence between distamycin binding (determined by footprinting analysis) and topoisomerase II-mediated cleavage of SV40 DNA (determined by sequencing gel analysis). Distamycin binding potentiated the cleavage of specific sites in the near proximity of distamycin-binding sites (within at least 25 base pairs), which indicates that DNA secondary structure is involved in topoisomerase II-DNA interactions. That distamycin potentiated cleavage only at sites that were recognized in the absence of distamycin and suppressed cleavage directly at distamycin-binding sites indicates that topoisomerase II recognizes DNA on the basis of primary sequence. In addition, distamycin stimulated topoisomerase II-mediated DNA relaxation and antagonized the inhibitory effect of VM-26. These results show that the DNA sequence-specific binding of distamycin produces local and propagated effects in the DNA which markedly affect topoisomerase II activity.     

DNA topoisomerases as targets for the anticancer drug TAS-103: primary cellular target and DNA cleavage enhancement

TAS-103 is a novel antineoplastic agent that is active against in vivo tumor models [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. This drug is believed to be a dual topoisomerase I/II-targeted agent, because it enhances both topoisomerase I- and topoisomerase II-mediated DNA cleavage in treated cells. However, the relative importance of these two enzymes for the cytotoxic actions of TAS-103 is not known. Therefore, the primary cellular target of the drug and its mode of action were determined. TAS-103 stimulated DNA cleavage mediated by mammalian topoisomerase I and human topoisomerase IIalpha and beta in vitro. The drug was less active than camptothecin against the type I enzyme but was equipotent to etoposide against topoisomerase IIalpha. A yeast genetic system that allowed manipulation of topoisomerase activity and drug sensitivity was used to determine the contributions of topoisomerase I and II to drug cytotoxicity. Results indicate that topoisomerase II is the primary cellular target of TAS-103. In addition, TAS-103 binds to human topoisomerase IIalpha in the absence of DNA, suggesting that enzyme-drug interactions play a role in formation of the ternary topoisomerase II.drug.DNA complex. TAS-103 induced topoisomerase II-mediated DNA cleavage at sites similar to those observed in the presence of etoposide. Like etoposide, it enhanced cleavage primarily by inhibiting the religation reaction of the enzyme. Based on these findings, it is suggested that TAS-103 be classified as a topoisomerase II-targeted drug.     

Local sequence requirements for DNA cleavage by mammalian topoisomerase II in the presence of doxorubicin

Doxorubicin, a DNA-intercalator, is one of several anti-cancer drugs that have been found to stabilizes topoisomerase II cleavage complexes at drug-specific DNA sites. The distribution and DNA sequence environments of doxorubicin-stabilized sites were determined in the SV40 genome. The sites were found to be most concentrated in the major nuclear matrix-associated region and nearly absent in the vicinity of the replication origin including the enhancer sequences in the 21-bp and 72-bp tandem repeats. Among 97 doxorubicin-stabilized sites that were localized at the DNA sequence level, none coincided with any of the 90 topoisomerase II cleavage sites detected in the same regions in the absence of drug. Cleavage at the 90 enzyme-only sites was inhibited by doxorubicin and never stimulated even at low drug concentrations. All of the doxorubicin-stabilized sites had an A at the 3' terminus of at least one member of each pair of strand breaks that would constitute a topoisomerase II double-strand scission. Conversely, none of the enzyme-only sites had an A simultaneously at the corresponding positions on opposite strands. The 3'-A requirement for doxorubicin-stabilized cleavage is therefore incompatible with enzyme-only cleavage and explains the mutual exclusivity of the two classes of sites.     

Base mutation analysis of topoisomerase II-idarubicin-DNA ternary complex formation. Evidence for enzyme subunit cooperativity in DNA cleavage.

Antitumor drugs, such as anthracyclines, interfere with mammalian DNA topoisomerase II by forming a ternary complex, DNA-drug-enzyme, in which DNA strands are cleaved and covalently linked to the enzyme. In this work, a synthetic 36-bp DNA oligomer derived from SV40 and mutated variants were used to determine the effects of base mutations on DNA cleavage levels produced by murine topoisomerase II with and without idarubicin. Although site competition could affect cleavage levels, mutation effects were rather similar among several cleavage sites. The major sequence determinants of topoisomerase II DNA cleavage without drugs are up to five base pairs apart from the strand cut, suggesting that DNA protein contacts involving these bases are particularly critical for DNA site recognition. Cleavage sites with adenines at positions -1 were detected without idarubicin only under conditions favouring enzyme binding to DNA, showing that these sites are low affinity sites for topoisomerase II DNA cleavage and/or binding. Moreover, the results indicated that the sequence 5'-(A)TA/(A)-3' (the slash indicates the cleaved bond, parenthesis indicate conditioned preference) from -3 to +1 positions constitutes the complete base sequence preferred by anthracyclines. An important finding was that mutations that improve the fit to the above consensus on one strand can also increase cleavage on the opposite strand, suggesting that a drug molecule may effectively interact with one enzyme subunit only and trap the whole dimeric enzyme. These findings documented that DNA recognition by topoisomerase II may occur at one or the other strand, and not necessarily at both of them, and that the two subunits can act cooperatively to cleave a double helix.     

Position-specific effect of ribonucleotides on the cleavage activity of human topoisomerase II

Beyond the normal DNA transactions mediated by topoisomerase II, we have recently demonstrated that the cleavage activity of the two human topoisomerase II isoforms is several-fold stimulated if a ribonucleotide rather than a deoxyribonucleotide is present at the scissile phosphodiester in one strand of the substrate. Here we show that ribonucleotides exert a position-specific effect on topoisomerase II-mediated cleavage without altering the sequence specificity of the enzyme. Ribonucleotides located within the 4 bp cleavage stagger stimulate topoisomerase II-mediated cleavage, whereas ribonucleotides located outside the stagger in general have an inhibitory effect. Results obtained from competition experiments indicate that the position-specific effect of ribonucleotides on topoisomerase II activity is caused by altered substrate interaction. When cleavage is performed with substrates containing one ribonucleotide in both strands or several ribonucleotides in one strand the effect of the individual ribonucleotides on cleavage is not additive. Finally, although topoisomerase II recognizes substrates with longer stretches of ribonucleotides, an RNA/DNA hybrid where one strand is composed entirely of RNA is not cleaved by the enzyme. The positional effect of ribonucleotides on topoisomerase II-mediated cleavage shares many similarities to the positional effect exerted by either abasic sites or base mismatches, demonstrating a general influence of DNA imperfections on topoisomerase II activity.     

Intercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II

Many intercalative antitumor drugs have been shown to induce reversible protein-linked DNA breaks in cultured mammalian cells. Using purified mammalian DNA topoisomerase II, we have demonstrated that the antitumor drugs ellipticine and 2-methyl-9-hydroxyellipticine (2-Me-9-OH-E+) can produce reversible protein-linked DNA breaks in vitro. 2-Me-9-OH-E+ which is more cytotoxic toward L1210 cells and more active against experimental tumors than ellipticine is also more effective in stimulating DNA cleavage in vitro. Similar to the effect of 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) on topoisomerase II in vitro, the mechanism of DNA breakage induced by ellipticines is most likely due to the drug stabilization of a cleavable complex formed between topoisomerase II and DNA. Protein denaturant treatment of the cleavable complex results in DNA breakage and covalent linking of one topoisomerase II subunit to each 5'-end of the cleaved DNA. Cleavage sites on pBR322 DNA produced by ellipticine or 2-Me-9-OH-E+ treatment mapped at the same positions. However, many of these cleavage sites are distinctly different from those produced by the antitumor drug m-AMSA which also targets at topoisomerase II. Our results thus suggest that although mammalian DNA topoisomerase II may be a common target of these antitumor drugs, drug-DNA-topoisomerase interactions for different antitumor drugs may be different.     

DNA topoisomerase II cleaves at specific sites in the 5'' flanking region of c-fos proto-oncogenes in vitro.

We have analyzed 1 kb of cloned human c-fos sequence (-711 to +287) for calf thymus DNA topoisomerase II cleavage sites in vitro. Using the anti-tumor drug VP16 (demethylepipodophyllotoxin-beta-D-glucoside) with purified topoisomerase II, we identify twelve sites. Five sites are clustered around position -306 in a region that possesses enhancer-like properties. A second cluster of three sites is positioned 15 bp upstream of the TATA promoter element. With a HeLa nuclear extract as a source of topoisomerase II, a subset of cleavage sites is conserved within the two clusters. The cleavage sites in the enhancer-like element are conserved in the homologous region of the murine c-fos. These findings raise the possibility that topoisomerase II is involved in mediation of mitogen-induced c-fos expression.     

A consensus sequence for cleavage by vertebrate DNA topoisomerase II.

Topoisomerase II, purified from chicken erythrocytes, was reacted with a large number of different DNA fragments and cleavages were catalogued in the presence and absence of drugs that stabilize the cleavage intermediate. Cleavages were sequenced to derive a consensus for topoisomerase II that predicts catalytic sites. The consensus is: (sequence; see text) where N is any base and cleavage occurs at the indicated mark between -1 and +1. The consensus accurately predicts topoisomerase II sites in vitro. This consensus is not closely related to the Drosophila consensus sequence, but the two enzymes show some similarities in site recognition. Topoisomerase II purified from human placenta cleaves DNA sites that are essentially identical to the chicken enzyme, suggesting that vertebrate type II enzymes share a common catalytic sequence. Both viral and tissue specific enhancers contain sites sharing strong homology to the consensus and endogenous topoisomerase II recognizes some of these sites in vivo.     

Site-specific DNA cleavage by Chlorella virus topoisomerase II

The DNA cleavage reaction of topoisomerase II is central to the catalytic activity of the enzyme and is the target for a number of important anticancer drugs. Unfortunately, efforts to characterize this fundamental reaction have been limited by the low levels of DNA breaks normally generated by the enzyme. Recently, however, a type II topoisomerase with an extraordinarily high intrinsic DNA cleavage activity was isolated from Chlorella virus PBCV-1. To further our understanding of this enzyme, the present study characterized the site-specific DNA cleavage reaction of PBCV-1 topoisomerase II. Results indicate that the viral enzyme cleaves DNA at a limited number of sites. The DNA cleavage site utilization of PBCV-1 topoisomerase II is remarkably similar to that of human topoisomerase IIalpha, but the viral enzyme cleaves these sites to a far greater extent. Finally, PBCV-1 topoisomerase II displays a modest sensitivity to anticancer drugs and DNA damage in a site-specific manner. These findings suggest that PBCV-1 topoisomerase II represents a unique model with which to dissect the DNA cleavage reaction of eukaryotic type II topoisomerases.     

Distribution of topoisomerase II cleavage sites in simian virus 40 DNA and the effects of drugs.

The distributions of DNA cleavage sites induced by topoisomerase II in the presence or absence of specific drugs were mapped in the simian virus 40 genome. The drugs studied were 5-iminodaunorubicin, amsacrine (m-AMSA), teniposide (VM-26) and 2-methyl-9-hydroxyellipticinium; each produced a distinctive pattern of enhanced cleavage. Consistently intense cleavage, both in the presence and in the absence of drugs, occurred in the nuclear matrix-associated region. Since topoisomerase II is a major constituent of the nuclear matrix, and cleavage complexes include a covalent link between topoisomerase II and DNA, the findings suggest that topoisomerase II may function to attach DNA to the nuclear matrix. Cleavage usually occurred on both DNA strands with the expected four base-pair 5' stagger, and strong sites tended to occur within A/T runs such as have been associated with binding to the nuclear scaffold. Intense cleavage was present also in the replication termination region, but was absent from the vicinity of the replication origin. Cleavage intensities were found to change with time in a manner that depended both on the site and on the drug, suggesting that topoisomerase II can move along the DNA from a kinetically preferred site to a thermodynamically preferred site.     

Effects of antineoplastic drugs on the post-strand-passage DNA cleavage/religation equilibrium of topoisomerase II.

The post-strand-passage DNA cleavage/religation equilibrium of Drosophila melanogaster topoisomerase II was examined. This was accomplished by including adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analogue which supports strand passage but not enzyme turnover, in assays. Levels of post-strand-passage enzyme-mediated DNA breakage were 3-5 times higher than those generated by topoisomerase II prior to the strand-passage event. This finding correlated with a decrease in the apparent first-order rate of topoisomerase II mediated DNA religation in the post-strand-passage cleavage complex. Since previous studies demonstrated that antineoplastic drugs stabilize the pre-strand-passage cleavage complex of topoisomerase II by impairing the enzyme's ability to religate cleaved DNA [Osheroff, N. (1989) Biochemistry 28, 6157-6160; Robinson, M.J., & Osheroff, N. (1990) Biochemistry 29, 2511-2515], the effects of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide on the enzyme's post-strand-passage DNA cleavage complex were characterized. Both drugs stimulated the ability of topoisomerase II to break double-stranded DNA after strand passage. As determined by two independent assay systems, m-AMSA and etoposide stabilized the enzyme's post-strand-passage DNA cleavage complex primarily by inhibiting DNA religation. These results strongly suggest that both the pre- and post-strand-passage DNA cleavage complexes of topoisomerase II serve as physiological targets for these structurally disparate antineoplastic drugs.     

N-acetyl-p-benzoquinone imine, the toxic metabolite of acetaminophen, is a topoisomerase II poison

Although acetaminophen is the most widely used analgesic in the world, it is also a leading cause of toxic drug overdoses. Beyond normal therapeutic doses, the drug is hepatotoxic and genotoxic. All of the harmful effects of acetaminophen have been attributed to the production of its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Since many of the cytotoxic/genotoxic events triggered by NAPQI are consistent with the actions of topoisomerase II-targeted drugs, the effects of this metabolite on human topoisomerase IIalpha were examined. NAPQI was a strong topoisomerase II poison and increased levels of enzyme-mediated DNA cleavage >5-fold at 100 microM. The compound induced scission at a number of DNA sites that were similar to those observed in the presence of the topoisomerase II-targeted anticancer drug etoposide; however, the relative site utilization differed. NAPQI strongly impaired the ability of topoisomerase IIalpha to reseal cleaved DNA molecules, suggesting that inhibition of DNA religation is the primary mechanism underlying cleavage enhancement. In addition to its effects in purified systems, NAPQI appeared to increase levels of DNA scission mediated by human topoisomerase IIalpha in cultured CEM leukemia cells. In contrast, acetaminophen did not significantly affect the DNA cleavage activity of the human enzyme in vitro or in cultured CEM cells. Furthermore, the analgesic did not interfere with the actions of etoposide against the type II enzyme. These results suggest that at least some of the cytotoxic/genotoxic effects caused by acetaminophen overdose may be mediated by the actions of NAPQI as a topoisomerase II poison.     

Application of a degenerate consensus sequence to quantify recognition sites by vertebrate DNA topoisomerase II

A consensus sequence has been derived for vertebrate topoisomerase II cleavage of DNA (Spitzner, J. R. and Muller, M. T. (1988) Nucleic Acid. Res. 16, 5533-5556). An independent sample of 65 topoisomerase II sites (obtained in the absence of topoisomerase II inhibitors) was analyzed and found to match the consensus sequence as well as enzyme sites determined in the presence of the anti-tumor drug 4'-(9-acridinyl-amino)-methanesulfon-m-anisidide (m-AMSA). As originally described, conventional application of the consensus sequence afforded accuracy in the prediction of the locations but not the frequencies of topoisomerase II cleavages. In the present report, we describe a new method which quantitatively discriminates sites from nonsites, called the 'matrix mean' method (the mean match of a site to the matrix of base proportions from the original consensus sequence derivation). Furthermore, we derived a second method, called the 'unique score' model, which predicts frequency of topoisomerase II activity at a cleavage site. In the unique score method both DNA strands of a site are examined to determine the total number of the consensus positions that match on at least one strand of a potential site. From the new data base of 65 topoisomerase II sites, cleavages were scored for relative cleavage strength. Linear regression analysis showed a significant (p less than 0.01) correlation between the unique score and cleavage strength. The study was extended to show that the unique score model accurately and quantitatively predicts topoisomerase II sites either in the absence or presence of m-AMSA using the same consensus sequence.