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.     

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.     

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.     

Novel partitioning of DNA cleavage sites for Drosophila topoisomerase II

We have examined the long-range distribution of double-stranded DNA cleavage sites for Drosophila melanogaster topoisomerase II. These studies reveal a novel partitioning of preferred topoisomerase II cleavage sites. In the eukaryotic DNAs examined, major cleavage sites were typically found in nontranscribed spacer segments and close to the 5' and 3' boundaries of genes. In contrast, there were few if any prominent cleavage sites within genes. In addition, most of the major topoisomerase II cleavage sites closely corresponded to naked DNA hypersensitive sites for the prokaryotic enzyme, micrococcal nuclease.     

Single strand DNA cleavage reaction of duplex DNA by Drosophila topoisomerase II

A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.     

Structure of the Drosophila DNA topoisomerase II gene. Nucleotide sequence and homology among topoisomerases II

We have determined the nucleotide sequence of the Drosophila DNA topoisomerase II gene. Data from primer extension and S1 nuclease protection experiments were combined with comparisons of genomic and cDNA sequences to determine the structure of the mature messenger RNA. This message has a large open reading frame of 4341 nucleotides. The length of the predicted protein is 1447 amino acids with a molecular weight of 164,424. Topoisomerase II can be divided into three domains: (1) an N-terminal region with homology to the B (ATPase) subunit of the bacterial type II topoisomerase, DNA gyrase; (2) a central region with homology to the A (breaking and rejoining) subunit of DNA gyrase; (3) a C-terminal region characterized by alternating stretches of positively and negatively charged amino acids. DNA topoisomerase II from the fruit fly shares significant sequence homology with those from divergent sources, including bacteria, bacteriophage T4 and yeasts. The location and distribution of homologous stretches in these sequences are analyzed.     

Conformational properties of topoisomerase II inhibitors and sequence specificity of DNA cleavage

The sequence specificity of topoisomerase-II-mediated DNA cleavage, stimulated by 2-methyl-9-hydroxy ellipticinium and 4′, 5′,7-trihydroyflavone (genistein) was investigated by sequencing analysis of DNA cleavage sites and molecular modeling techniques. The former drug exhibits a marked preference for a T base at the position immediately preceding the cleavage site (?1). The latter shares the preference for the same base, with an additional preference for a thymine at position +1. The cleavage intensity patterns for the two drugs differ considerably. From a conformational point of view, ellipticinium and genistein exhibit similar overall shape and dimensions. However, the fused ring system in the former generates a planar structure whereas the single bond, connecting the two aromatic portions in the latter, allows internal rotation. The most stable conformation of genistein corresponds to a deviation of about 40° from planarity. A computer-assisted analysis was carried out to compare the steric and electrostatic properties of the two compounds. Two types of preferred (energetically almost degenerate) alignment for the two molecules were found. One corresponds to overlapping of the 9-hydroxyl containing ring of ellipticinium with the 4′-hydroxyphenyl moiety of genistein, the other envisages the same moiety of ellipticine superimposed to the hydroxyl-benzopyrone portion of genistein. The structural similarities of the test drugs might account for the common preference for stimulation of DNA cleavage at position +1, whereas the different possible arrangements of genistein in the cleavable complex could explain both the additional +1 specificity exhibited by this compound and the differences in cleavage intensity patterns observed in comparison to ellipticinium.     

In vivo mapping of DNA topoisomerase II-specific cleavage sites on SV40 chromatin

The antitumor drug, m-AMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidide), is known to interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by blocking the enzyme-DNA complex in its putative cleavable state. Treatment of SV40 virus infected monkey cells with m-AMSA resulted in both single- and double-stranded breaks on SV40 viral chromatin. These strand breaks are unusual because they are covalently associated with protein. Immunoprecipitation results suggest that the covalently linked protein is DNA topoisomerase II. These results are consistent with the proposal that the drug action in vivo involves the stabilization of a cleavable complex between topoisomerase II and DNA in chromatin. Mapping of these double-stranded breaks on SV40 viral DNA revealed multiple topoisomerase II cleavage sites. A major topoisomerase II cleavage site was preferentially induced during late infection and was mapped in the DNAase I hypersensitive region of SV40 chromatin.     

8-methoxycaffeine inhibition of Drosophila DNA topoisomerase II.

We have investigated the effect of 8-methoxycaffeine on the interaction between Drosophila DNA topoisomerase II and DNA. We have shown that 8-methoxycaffeine affected the enzyme strand-passing activity by inhibiting decatenation of kinetoplast DNA, and that it interfered with the breakage-reunion reaction by stabilizing a cleavable complex. Treatment of the cleavable complex with protein denaturant resulted in DNA breaks. High resolution mapping of the cleavage sites in the central spacer region of Tetrahymena rDNA revealed that, contrary to what was observed with clinically important DNA topoisomerase II inhibitors, 8-methoxycaffeine did not modify the cleavage pattern observed without the drug.     

Eukaryotic topoisomerase II cleavage of parallel stranded DNA tetraplexes.

A guanine-rich single-stranded DNA from the human immunoglobulin switch region was shown by Sen and Gilbert [Nature, (1988) 334, 364-366] to be able to self-associate to form a stable four-stranded parallel DNA structure. Topoisomerase II did not cleave the single-stranded DNA molecule. Surprisingly, the enzyme did cleave the same DNA sequence when it was annealed into the four-stranded structure. The two cleavage sites observed were the same as those found when this DNA molecule was paired with a complementary molecule to create a normal B-DNA duplex. These cleavages were shown to be protein-linked and reversible by the addition of salt, suggesting a normal topoisomerase II reaction mechanism. In addition, an eight-stranded DNA molecule created by the association of a complementary oligonucleotide with the four-stranded structure was also cleaved by topoisomerase II despite being resistant to restriction endonuclease digestion. These results suggest that a single strand of DNA may possess the sequence information to direct topoisomerase II to a binding site, but the site must be base paired in a proper manner to do so. This demonstration of the ability of a four-stranded DNA molecule to be a substrate for an enzyme further suggests that these DNA structures may be present in cells.     

In vivo inhibition of trypanosome mitochondrial topoisomerase II: effects on kinetoplast DNA maxicircles.

Kinetoplast DNA, the mitochondrial DNA of trypanosomes, is a topologically complex structure composed of interlocked minicircles and maxicircles. We previously reported that etoposide, a potent inhibitor of topoisomerase II, promotes the cleavage of about 20% of network minicircle DNA (T. A. Shapiro, V. A. Klein, and P. T. Englund, J. Biol. Chem. 264:4173-4178, 1989). We now find that virtually all maxicircles are released from kinetoplast DNA networks after trypanosomes are treated with etoposide. As expected for a topoisomerase II cleavage product, the linearized maxicircles have protein bound to both 5' ends. After etoposide treatment, the residual minicircle catenanes have a sedimentation coefficient which is only 70% that of controls, and by electron microscopy the networks are less compact. Double-size networks, the characteristic dumbbell-shape forms that normally arise in the final stages of network replication, are replaced by aberrant unit-size forms.     

DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA

In order to study the double-strand DNA passage reaction of eukaryotic type II topoisomerases, a quantitative assay to monitor the enzymic conversion of supercoiled circular DNA to relaxed circular DNA was developed. Under conditions of maximal activity, relaxation catalyzed by the Drosophila melanogaster topoisomerase II was processive and the energy of activation was 14.3 kcal . mol-1. Removal of supercoils was accompanied by the hydrolysis of either ATP or dATP to inorganic phosphate and the corresponding nucleoside diphosphate. Apparent Km values were 200 microM for pBR322 plasmid DNA, 140 microM for SV40 viral DNA, 280 microM for ATP, and 630 microM for dATP. The turnover number for the Drosophila enzyme was at least 200 supercoils of DNA relaxed/min/molecule of topoisomerase II. The enzyme interacts preferentially with negatively supercoiled DNA over relaxed molecules, is capable of removing positive superhelical twists, and was found to be strongly inhibited by single-stranded DNA. Kinetic and inhibition studies indicated that the beta and gamma phosphate groups, the 2'-OH of the ribose sugar, and the C6-NH2 of the adenine ring are important for the interaction of ATP with the enzyme. While the binding of ATP to Drosophila topoisomerase II was sufficient to induce a DNA strand passage event, hydrolysis was required for enzyme turnover. The ATPase activity of the topoisomerase was stimulated 17-fold by the presence of negatively supercoiled DNA and approximately 4 molecules of ATP were hydrolyzed/supercoil removed. Finally, a kinetic model describing the switch from a processive to a distributive relaxation reaction is presented.     

Developmental regulation of Drosophila DNA topoisomerase II

Affinity-purified polyclonal antibodies were used to quantitate steady-state levels of DNA topoisomerase II (topo II) throughout Drosophila development. Although wide fluctuations were recorded at different stages, these fluctuations were paralleled by changes in levels of the nuclear lamin, a nuclear structural protein used as an internal standard. The exception to this was adult males where lamin levels were significantly elevated relative to topo II. Northern blot analyses of topo II and lamin mRNA, performed in conjunction with immunoblot analyses of protein revealed fluctuations in levels of the two different messages that paralleled changes in each other and in their respective translation products. Biochemical and immunochemical analyses were complemented by indirect immunofluorescence and immunoperoxidase experiments performed in situ. topo II was found distributed throughout nuclei in most but not all cell types examined. These results for Drosophila topo II are apparently at odds with those obtained by others working in vertebrate systems (see, for example, Heck, M. M. S. and W. C. Earnshaw. 1986. J. Cell Biol. 103:2569-2581; Heck, M. M. S., W. N. Hittelman, and W. C. Earnshaw. 1988. Proc. Natl. Acad. Sci. USA. 85:1086-1090) and suggest that in Drosophila, topo II may not be a useful marker for the proliferative state.     

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.     

Thioguanine substitution alters DNA cleavage mediated by topoisomerase II.

Thiopurines and topoisomerase II-targeted drugs (e.g., etoposide) are widely used anticancer drugs. However, topoisomerase II-targeted drugs can cause acute myeloid leukemia, with the risk of this secondary leukemia linked to a genetic defect in thiopurine catabolism. Chronic thiopurines result in thioguanine substitution in DNA. The effect of these substitutions on DNA topoisomerase II activity is not known. Our goal was to determine whether deoxythioguanosine substitution alters DNA cleavage stabilized by human topoisomerase II. We studied four variations of a 40 mer oligonucleotide with a topoisomerase II cleavage site, each with a single deoxythioguanosine in a different position relative to the cleavage site (-1 or +2 in the top and +2 or +4 in the bottom strand). Deoxythioguanosine substitution caused position-dependent quantitative effects on cleavage. With the -1 or +2 top and +2 or +4 bottom substitutions, mean topoisomerase II-induced cleavage was 0.6-, 2.0-, 1.1-, and 3.3-fold that with the wild-type substrate (P=0. 011, < 0.008, 0.51, and < 0.001, respectively). In the presence of 100 microM etoposide, cleavage was enhanced for wild-type and all thioguanosine-modified substrates relative to no etoposide, with the +4 bottom substitution showing greater etoposide-induced cleavage than the wild-type substrate (P=0.015). We conclude that thioguanine incorporation alters the DNA cleavage induced by topoisomerase II in the presence and absence of etoposide, providing new insights to the mechanism of thiopurine effect and on the leukemogenesis of thiopurines, with or without topoisomerase inhibitors.     

The nucleotide sequence of the fission yeast DNA topoisomerase II gene: structural and functional relationships to other DNA topoisomerases.

We have determined the complete nucleotide sequence of a 5.3-kb long genomic DNA fragment of the fission yeast Schizosaccharomyces pombe that encodes DNA topoisomerase II. It contains a 4293 bp long single open reading frame. The predicted polypeptide has 1431 residues (mol. wt 162,000) and shows three characteristic domains; the large C-terminal region, which consists of alternating acidic-basic stretches and might be a chromatin-binding domain, the NH2 half domain homologous to the ATP-binding gyrB subunit of bacterial gyrase and the central-to-latter part which is homologous to the NH2 domain of the catalytic gyrA subunit, suggesting a possible evolutionary consequence of the gene fusion of the bacterial gyrase subunits into the eucaryotic DNA topoisomerase II gene. We have found that the cloned fission yeast TOP2 gene can complement the budding yeast top2 mutation, although the fission yeast TOP2 protein sequence is only 50% homologous to the recently determined sequence of budding yeast (J.C. Wang, personal communication). Conversely, the budding yeast TOP2 gene can complement the fission yeast top2 mutations, indicating that their DNA topoisomerase II genes are functionally exchangeable.     

Distribution of topoisomerase II-mediated cleavage sites and relation to structural and functional landmarks in 830 kb of Drosophila DNA.

The pattern of sites for cleavage mediated by topoisomerase II was determined in 830 kb of cloned DNA from the Drosophila X chromosome, with the objectives of comparing it with mapped structural and functional landmarks and examining if the correlations with such landmarks reported in individual loci can be generalized to a region approximately 100 times longer. The relative frequencies of topoisomerase II cleavage sites in 247 restriction fragments from 67 clones were quantified by hybridization with probes prepared from DNA fragments which abutted all cleavage sites in each clone, selected through the covalently bound topoisomerase II subunit; the specificity and quantitative nature of this method were demonstrated using a plasmid DNA model. The 12 restriction fragments with strong nuclear scaffold attachment (SAR) activity, of which seven possess autonomous replication (ARS) activity, show statistically strong coincidence or contiguity ( P </=0.11) with regions of high topoisomerase II cleavage site frequency. These regions show no correlation with repetitive sequence or A/T or C/G content and some extend over >10 kb; their sensitivity is therefore unlikely to be due to alternating purine-pyrimidine repeats or regions of Z conformation, which are preferred motifs. The hypothesis that they possess intrinsic curvature is consistent with the similarity of their length and spacing to regions of predicted curvature in the 315 kb DNA of Saccharomyces cerevisiae chromosome III and with the reported strong binding preference of topoisomerase II for curved DNA. The topoisomerase II cleavage pattern in this DNA further shows that its relationships to functional properties seen in individual loci, especially to MAR/SAR and ARS activity and to the restricted accessibility of DNA to topoisomerase II in vivo, can be generalized to much longer regions of the genome.