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.     

Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.     

DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.     

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.     

Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I

Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently. Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA. These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria. Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature. Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation. Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission. Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies. The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein. Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions. This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme. Noncovalent binding is therefore independent of competence in transesterification. It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes.     

Homocamptothecin, an E-ring-modified camptothecin analogue, generates new topoisomerase I-mediated DNA breaks

Homocamptothecin (hCPT) contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone ring found in camptothecin and its tumor active analogues, including topotecan and irinotecan. The homologation of the lactone E-ring reinforces the stability of the lactone, thus reducing considerably its conversion into a carboxylate form which is inactive. We have recently shown that hCPT is much more active than the parent compound against a variety of tumor cells in vitro and in xenograft models, suggesting that a highly reactive lactone is not essential for topoisomerase I-mediated anticancer activity [Lesueur-Ginot et al. (1999) Cancer Res. 59, 2939-2943]. In the present study, we provide further evidence that hCPT has superior topoisomerase I inhibition capacities to CPT. In particular, we show that replacement of the camptothecin lactone E-ring with a homologous seven-membered lactone ring changes the sequence-specificity of the drug-induced DNA cleavage by topoisomerase I. Both CPT and hCPT stimulate the cleavage by topoisomerase I at T( downward arrow)G sites, but in addition, hCPT stabilizes cleavage at specific sites containing the sequence AAC( downward arrow)G. At low drug concentrations, the cleavage at the T( downward arrow)G sites and at the hCPT-specific C( downward arrow)G sites is more pronounced and more stable with hCPT than with CPT. The in vitro data were confirmed in cells. Higher levels of protein-DNA complexes were detected in P388 leukemia cells treated with hCPT than those treated with CPT. Immunoblotting experiments revealed that endogenous topoisomerase I was efficiently trapped onto DNA by hCPT in cells. Finally, the use of a leukemia cell line resistant to CPT provided evidence that topoisomerase I is involved in the cytotoxicity of hCPT. Altogether, the results show that the beta-hydroxylactone ring of hCPT plays an important and positive role in the poisoning of topoisomerase I. An explanation is proposed to account for such remarkable changes in the sequence specificity of topoisomerase I cleavage consequent to the modification of the lactone. The study sheds new light on the importance of the lactone ring of camptothecins for the stabilization of topoisomerase I-DNA complexes.     

The geometry of DNA supercoils modulates topoisomerase-mediated DNA cleavage and enzyme response to anticancer drugs

Collisions with DNA tracking systems are critical for the conversion of transient topoisomerase-DNA cleavage complexes to permanent strand breaks. Since DNA is overwound ahead of tracking systems, cleavage complexes most likely to produce permanent strand breaks should be formed between topoisomerases and positively supercoiled molecules. Therefore, the ability of human topoisomerase IIalpha and IIbeta and topoisomerase I to cleave positively supercoiled DNA was assessed in the absence or presence of anticancer drugs. Topoisomerase IIalpha and IIbeta maintained approximately 4-fold lower levels of cleavage complexes with positively rather than negatively supercoiled DNA. Topoisomerase IIalpha also displayed lower levels of cleavage with overwound substrates in the presence of nonintercalative drugs. Decreased drug efficacy was due primarily to a drop in baseline (i.e., nondrug) cleavage, rather than an altered interaction with the enzyme-DNA complex. Similar results were seen for topoisomerase IIbeta, but the effects of DNA geometry on drug-induced scission were somewhat less pronounced. With both topoisomerase IIalpha and IIbeta, intercalative drugs displayed greater relative cleavage enhancement with positively supercoiled DNA. This appeared to result from negative effects of high concentrations of intercalative agents on underwound DNA. In contrast to the type II enzymes, topoisomerase I maintained approximately 3-fold higher levels of cleavage complexes with positively supercoiled substrates and displayed an even more dramatic increase in the presence of camptothecin. These findings suggest that the geometry of DNA supercoils has a profound influence on topoisomerase-mediated DNA scission and that topoisomerase I may be an intrinsically more lethal target for anticancer drugs than either topoisomerase IIalpha or IIbeta.     

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.     

Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide.

Several classes of antitumor drugs are known to stabilize topoisomerase complexes in which the enzyme is covalently bound to a terminus of a DNA strand break. The DNA cleavage sites generally are different for each class of drugs. We have determined the DNA sequence locations of a large number of drug-stimulated cleavage sites of topoisomerase II, and find that the results provide a clue to the possible structure of the complexes and the origin of the drug-specific differences. Cleavage enhancements by VM-26 and amsacrine (m-AMSA), which are representative of different classes of topoisomerase II inhibitors, have strong dependence on bases directly at the sites of cleavage. The preferred bases were C at the 3' terminus for VM-26 and A at the 5' terminus for m-AMSA. Also, a region of dyad symmetry of 12 to 16 base pairs was detected about the enzyme cleavage positions. These results are consistent with those obtained with doxorubicin, although in the case of doxorubicin, cleavage requires the presence of an A at the 3' terminus of at least one the pair of breaks that constitute a double-strand cleavage (Capranico et al., Nucleic Acids Res., 1990, 18: 6611). These findings suggest that topoisomerase II inhibitors may stack with one or the other base pair flanking the enzyme cleavage sites.     

Sequence requirements for mammalian topoisomerase II mediated DNA cleavage stimulated by an ellipticine derivative.

Various antitumor drugs stabilize DNA topoisomerase II-DNA transient covalent complexes. The complexes distribution along pBR322 DNA was shown previously to depend upon the nature of the drug (Tewey et al. (1984) Science 226, 466-468). The position in pBR322 of DNA cleavage by calf DNA topoisomerase II for 115 such sites stabilized by an ellipticine derivative and the relative frequency of cleavage at most of these sites were determined. The nucleotide sequence surrounding the 25 strongest sites was analyzed and the following ellipticine specific consensus sequence was deduced: 5'-ANCNT(A/G)T.NN(G/C)N(A/G)-3' where cleavage occurs at the indicated mark. A thymine is always present at the 3' end of at least one strand of the strong cleavage sites, and the dinucleotide AT or GT at the 3' end of the break plays a major role in the complex stabilisation. The predictive value of cleavage of the consensus was tested for two regions of SV40 DNA and cleavage was indeed detected at the majority of the sites matching the consensus. Some complexes stabilized by ellipticine are resistant to salt dissociation and this property seems to be correlated with the presence of symmetrical sequences in the cleavage site with a center of symmetry staggered relatively to the center of symmetry of cleavage.     

Interactions of Drosophila DNA topoisomerase II with left-handed Z-DNA in supercoiled minicircles.

The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families of mini and mini-CG were studied by band-shift gel electrophoresis in which the individual topoisomers and their discrete or aggregated protein complexes were resolved. A monoclonal anti-Z-DNA IgG antibody (23B6) bound and aggregated only mini-CG, thereby confirming the presence of Z-DNA. Topoisomerase II bound and relaxed mini-CG more readily than mini. In both cases, there was a preference for more highly negatively supercoiled topoisomers. The topoisomerase II inhibitor VM-26 induced the formation of stable covalent DNA-protein intermediates. In addition, the non-hydrolyzable GTP analogue GTP gamma S inhibited the binding and relaxation activities. Experiments to detect topoisomerase cleavage sites failed to elicit specific loci on either minicircle preparation. We conclude that Drosophila topoisomerase II is able to bind and process small minicircles with lengths as short as 360 bp and negative superhelix densities, - sigma, which can exceed 0.1. Furthermore, the enzyme has a preferential affinity for topoisomers containing Z-DNA segments and relaxes these molecules, presumably by cleavage external to the inserts. Thus, a potentially functional relationship between topoisomerase II, an enzyme regulating the topological state of DNA-chromatin in vivo, and left-handed Z-DNA, a conformation stabilized by negative supercoiling, has been established.     

Drosophila topoisomerase II double-strand DNA cleavage: analysis of DNA sequence homology at the cleavage site.

In order to study the sequence specificity of double-strand DNA cleavage by Drosophila topoisomerase II, we have mapped and sequenced 16 strong and 47 weak cleavage sites in the recombinant plasmid p pi 25.1. Analysis of the nucleotide and dinucleotide frequencies in the region near the site of phosphodiester bond breakage revealed a nonrandom distribution. The nucleotide frequencies observed would occur by chance with a probability less than 0.05. The consensus sequence we derived is 5'GT.A/TAY decrease ATT.AT..G 3', where a dot means no preferred nucleotide, Y is for pyrimidine, and the arrow shows the point of bond cleavage. On average, strong sites match the consensus better than weak sites.     

Characterization of a camptothecin-resistant human DNA topoisomerase I

Topoisomerase I purified from a camptothecin-resistant human leukemia cell line and from the parental, camptothecin-sensitive line were compared in vitro. Relaxation of supercoiled DNA by the wild type enzyme was inhibited in the presence of camptothecin, while the mutant enzyme was unimpaired. Camptothecin altered the cleavage pattern of the wild type but not of the mutant enzyme. The stability of cleavable complexes was studied at a preferred topoisomerase I-binding sequence recognized by both enzymes. Camptothecin greatly enhanced the kinetic stability of the cleavable complex formed by the wild type enzyme, whereas that of the mutant enzyme was only marginally affected. In the absence of camptothecin, the cleavable complex formed by the mutant enzyme was stabilized relative to that of the wild type by several criteria. Thus, the mutant enzyme cleaved the topoisomerase I recognition sequence with 2-fold higher efficiency than the wild type enzyme. The mutant cleavable complex had a higher kinetic stability and was less sensitive to salt dissociation than the wild type complex. Furthermore, the mutant enzyme formed cleavable complexes in the absence of divalent cations, which were required for complex formation by the wild type enzyme.     

Stimulation of topoisomerase II mediated DNA cleavage at specific sequence elements by the 2-nitroimidazole Ro 15-0216

The effect of the 2-nitroimidazole Ro 15-0216 upon the interaction between purified topoisomerase II and its DNA substrate was investigated. The cleavage reaction in the presence of this DNA-nonintercalative drug took place with the hallmarks of a regular topoisomerase II mediated cleavage reaction, including covalent linkage of the enzyme to the cleaved DNA. In the presence of Ro 15-0216, topoisomerase II mediated cleavage was extensively stimulated at major cleavage sites of which only one existed in the 4363 base pair pBR322 molecule. The sites stimulated by Ro 15-0216 shared a pronounced sequence homology, indicating that a specific nucleotide sequence is crucial for the action of this drug. The effect of Ro 15-0216 thus differs from that of the clinically important topoisomerase II targeted agents such as mAMSA, VM26, and VP16, which enhance enzyme-mediated cleavage at a multiple number of sites. In contrast to the previous described drugs, Ro 15-0216 did not exert any inhibitory effect on the enzyme's catalytic activity. This observation might be ascribed to the low stability of the cleavage complexes formed in the presence of Ro 15-0216 as compared to the stability of the ones formed in the presence of traditional topoisomerase II targeted drugs.     

Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites

A DNA consensus sequence for topoisomerase II cleavage sites was derived previously based on a statistical analysis of the nucleotide sequences around 16 sites that can be efficiently cleaved by Drosophila topoisomerase II (Sander, M., and Hsieh, T. (1985) Nucleic Acids Res. 13, 1057-1072). A synthetic 21-mer DNA sequence containing this cleavage consensus sequence was cloned into a plasmid vector, and DNA topoisomerase II can cleave this sequence at the position predicted by the cleavage consensus sequence. DNase I footprint analysis showed that topoisomerase II can protect a region of approximately 25 nucleotides in both strands of the duplex DNA, with the cleavage site located near the center of the protected region. Similar correlation between the DNase I footprints and strong topoisomerase II cleavage sites has been observed in the intergenic region of the divergent HSP70 genes. This analysis therefore suggests that the strong DNA cleavage sites of Drosophila topoisomerase II likely correspond to specific DNA-binding sites of this enzyme. Furthermore, the extent of DNA contacts made by this enzyme suggests that eucaryotic topoisomerase II, in contrast to bacterial DNA bacterial DNA gyrase, cannot form a complex with extensive DNA wrapping around the enzyme. The absence of DNA wrapping is probably the mechanistic basis for the lack of DNA supercoiling action for eucaryotic topoisomerase II.     

Overexpression of type I topoisomerases sensitizes yeast cells to DNA damage

DNA topoisomerases play essential roles in many DNA metabolic processes. It has been suggested that topoisomerases play an essential role in DNA repair. Topoisomerases can introduce DNA damage upon exposure to drugs that stabilize the covalent protein-DNA intermediate of the topoisomerase reaction. Lesions in DNA are also able to trap topoisomerase-DNA intermediates, suggesting that topoisomerases have the potential to either assist in DNA repair by locating sites of damage or exacerbating DNA damage by generation of additional damage at the site of a lesion. We have shown that overexpression of yeast topoisomerase I (TOP1) conferred hypersensitivity to methyl methanesulfonate and other DNA-damaging agents, whereas expression of a catalytically inactive enzyme did not. Overexpression of topoisomerase II did not change the sensitivity of cells to these DNA-damaging agents. Yeast cells lacking TOP1 were not more resistant to DNA damage than cells expressing wild type levels of the enzyme. Yeast topoisomerase I covalent complexes can be trapped efficiently on UV-damaged DNA. We suggest that TOP1 does not participate in the repair of DNA damage in yeast cells. However, the enzyme has the potential of exacerbating DNA damage by forming covalent DNA-protein complexes at sites of DNA damage.     

Protein:DNA interactions at chromosomal loop attachment sites

We have recently identified an evolutionarily conserved class of sequences that organize chromosomal loops in the interphase nucleus, which we have termed "matrix association regions" (MARs). MARs are about 200 bp long, AT-rich, contain topoisomerase II consensus sequences and other AT-rich sequence motifs, often reside near cis-acting regulatory sequences, and their binding sites are abundant (greater than 10,000 per mammalian nucleus). Here we demonstrate that the interactions between the mouse kappa immunoglobulin gene MAR and topoisomerase II or the "nuclear matrix" occur between multiple and sometimes overlapping binding sites. Interestingly, the sites most susceptible to topoisomerase II cleavage are localized near the breakpoints of a previously described illegitimate recombination event. The presence of multiple binding sites within single MARs may allow DNA and RNA polymerase passage without disrupting primary loop organization.     

Cleavage of DNA by mammalian DNA topoisomerase II

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.     

Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.