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PubMed1993

Meiosis-specific arrest revealed in DNA topoisomerase II mutants.

Although the processes of mitosis and meiosis are similar, there is evidence for fundamental regulatory differences between the two. To examine these differences, we have compared the meiotic phenotype of DNA topoisomerase II mutants with their previously described mitotic phenotype (C. Holm, T. Goto, J. Wang, and D. Botstein, Cell 41:553-563, 1985). top2 mutants in meiosis show no defects in the latest detectable stages of recombination, yet they arrest prior to spindle establishment at meiosis I. Fluorescence and electron microscopy reveal that top2 mutants exhibit wild-type levels of meiotic chromosome condensation and form morphologically normal synaptonemal complex but are delayed in the exit from pachytene. Arrested cells retain viability and form colonies if transferred to mitotic medium. Our results suggest that the top2 meiotic arrest is regulatory in nature. This arrest may have evolved to ensure the resolution of fortuitous tangles between nonhomologous chromosomes.

Chromosomes, Fungal↗
PubMed1997

Topoisomerase function during replication-independent chromatin assembly in yeast.

DNA topoisomerases I and II are the two major nuclear enzymes capable of relieving torsional strain in DNA. Of these enzymes, topoisomerase I plays the dominant role in relieving torsional strain during chromatin assembly in cell extracts from oocytes, eggs, and early embryos. We tested if the topoisomerases are used differentially during chromatin assembly in Saccharomyces cerevisiae by a combined biochemical and pharmacological approach. As measured by plasmid supercoiling, nucleosome deposition is severely impaired in assembly extracts from a yeast mutant with no topoisomerase I and a temperature-sensitive form of topoisomerase II (strain top1-top2). Expression of wild-type topoisomerase II in strain top1-top2 fully restored assembly-driven supercoiling, and assembly was equally efficient in extracts from strains expressing either topoisomerase I or II alone. Supercoiling in top1-top2 extract was rescued by adding back either purified topoisomerase I or II. Using the topoisomerase II poison VP-16, we show that topoisomerase II activity during chromatin assembly is the same in the presence and absence of topoisomerase I. We conclude that both topoisomerases I and II can provide the DNA relaxation activity required for efficient chromatin assembly in mitotically cycling yeast cells.

Chromatin↗
PubMed1999

Identification of cis and trans elements involved in the cell cycle regulation of multiple genes in Crithidia fasciculata.

Transcripts of several DNA replication genes, including the RPA1 and TOP2 genes, encoding the large subunit of nuclear replication protein A and the kinetoplast topoisomerase II, accumulate periodically during the cell cycle in the trypanosomatid Crithidia fasciculata. An octamer consensus sequence, CAUAGAAG, present in the 5' untranslated regions (UTR) of these mRNAs is required for periodic accumulation of the TOP2 and RPA1 transcripts and also for binding of a nuclear factor(s) to the 5' UTR RNAs of these genes. We show here that insertion of multiple (six) copies of this octamer sequence (6x octamer) into the 5' UTR of a reporter gene confers periodic accumulation on its transcript. Competition experiments and UV cross-linking studies show that the 6x octamer RNA and TOP2 5' UTR RNA bind to the same nuclear factor(s). Single-nucleotide substitutions in the 6x octamer that abolish the RNA gel shift also prevent cyclic accumulation of the reporter gene transcript. A protein termed cycling element binding protein, purified by affinity chromatography using 6x octamer RNA as a ligand, binds to RNAs containing wild-type octamers and not to those with mutant octamers. These results define a small sequence element in C. fasciculata mRNAs required for their cell cycle regulation and report the identification and purification of a putative regulatory protein that binds specifically to these elements.

5' Untranslated Regions↗
PubMed2006

Roles of nonhomologous end-joining pathways in surviving topoisomerase II-mediated DNA damage.

Topoisomerase II is a target for clinically active anticancer drugs. Drugs targeting these enzymes act by preventing the religation of enzyme-DNA covalent complexes leading to protein-DNA adducts that include single- and double-strand breaks. In mammalian cells, nonhomologous repair pathways are critical for repairing topoisomerase II-mediated DNA damage. Because topoisomerase II-targeting agents, such as etoposide, can also induce chromosomal translocations that can lead to secondary malignancies, understanding nonhomologous repair of topoisomerase II-mediated DNA damage may help to define strategies that limit this critical side effect on an important class of anticancer agents. Using Saccharomyces cerevisiae as a model eukaryote, we have determined the contribution of genes required for nonhomologous end-joining (NHEJ) for repairing DNA damage arising from treatment with topoisomerase II poisons, such as etoposide and 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA). To increase cellular sensitivity to topoisomerase II poisons, we overexpressed either wild-type or drug-hypersensitive alleles of yeast topoisomerase II. Using this approach, we found that yku70 (hdf1), yku80 (hdf2), and other genes required for NHEJ were important for cell survival following exposure to etoposide. The clearest increase in sensitivity was observed with cells overexpressing an etoposide-hypersensitive allele of TOP2 (Ser740Trp). Hypersensitivity was also seen in some end-joining defective mutants exposed to the intercalating agent mAMSA, although the increase in sensitivity was less pronounced. To confirm that the increase in sensitivity was not solely due to the elevated expression of TOP2 or due to specific effects of the drug-hypersensitive TOP2 alleles, we also found that deletion of genes required for NHEJ increased the sensitivity of rad52 deletions to both etoposide and mAMSA. Taken together, these results show a clear role for NHEJ in the repair of DNA damage induced by topoisomerase II-targeting agents and suggest that this pathway may participate in translocations generated by drugs, such as etoposide.

Amsacrine↗
PubMed2000

In vivo site specificity and human isoenzyme selectivity of two topoisomerase II-poisoning anthracyclines.

Anthracyclines exert antitumor activity by stimulating site-selective DNA cleavage by topoisomerase II (top2). DNA cleavage sites stimulated by two anthracycline analogues, dh-EPI and da-IDA, were investigated at the histone gene cluster of cultured Drosophila Kc cells. The two agents stimulated analogue-specific patterns of double-stranded DNA cleavage in Kc cell chromatin. Analyses of 47 base sequences of dh-EPI sites showed that the analogue largely followed the in vitro selectivity rule, the requirement of (5')TA at 3' ends of cleaved strands. da-IDA was more selective than dh-EPI, and thus fewer sites could be collected. Nevertheless, base sequences were consistent with its in vitro base preferences. DNA cleavage was then studied in vitro with Drosophila and human top2 isoforms. The tested drugs stimulated distinct in vitro patterns that corresponded to the in vivo patterns. Human top2alpha promoted cleavage patterns that were much more similar to those of Drosophila top2 (both in vitro and in vivo) than human top2beta. Moreover, da-IDA showed a marked site-dependent preference for human top2beta. Thus, DNA site selection in vivo is different for the test anthracyclines, and together with a degree of beta-form specificity, may affect drug activity in human cells.

Animals↗
PubMed1994

Saintopin, a dual inhibitor of DNA topoisomerases I and II, as a probe for drug-enzyme interactions.

Stabilization of the topoisomerase-cleavable complexes is the common initial event leading to the cytotoxicity of topoisomerase I and II (top1 and top2) inhibitors. Using saintopin (STP), a poison of both topoisomerases, we studied top1- and top2-cleavable complexes (Yamashita, Y., Kawada, S.-Z., Fujii, N., and Nakano, H. (1991) Biochemistry 30, 5838-5845). top1 and top2 sites induced in the presence of STP showed the same preferences for the base located 3' to the topoisomerase-induced DNA break (position +1): preference for G and not C. A camptothecin-resistant top1 with a mutation (Asn722-->Ser) next to the catalytic tyrosine (Tyr723) was cross-resistant to STP, suggesting that both STP and camptothecin interact with the protein near the catalytic tyrosine. These results are consistent with a dual interaction of the drug with the enzyme and the DNA and provide further evidence for the "drug-stacking" model. This model proposes that topoisomerase inhibitors bind, possibly through hydrogen bonding and/or stacking, with one of the bases flanking the DNA termini (guanine at position +1 in the case of STP) and within the enzyme catalytic pocket, most likely by stacking with the catalytic tyrosine.

Base Sequence↗
PubMed1986

Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis.

By examining cytological phenotypes of 587 temperature-sensitive mutants of the fission yeast Schizosaccharomyces pombe, we obtained 18 mutants which cause cell division in the absence of nuclear division. By genetic analyses, these novel nuclear division arrest mutants can be classified into nine complementation groups (designated cut1 - cut9). The cytological phenotype of cut mutants is similar but not identical to that of DNA topoisomerase II mutants (top2). The cut1 gene was cloned by transformation and shown to complement cut2 as well as cut1, indicating a functional relationship between the two genes. The cut genes are required for nuclear division, but their mutant phenotypes differ from most of the previously identified mutants which block nuclear division and also the subsequent cytokinesis. Fluorescence microscopy indicates that the mitotic chromosomes formed in cut mutant cells are abnormal and fail to separate properly. We suggest that cut mutations, like top2, block mitotic chromosome formation and concomitantly nuclear division, but that cytokinesis proceeds independently of the defects in nuclear division, demonstrating uncoordinated mitotic pathways. A novel mutant nuc1 is also described which shows a cytological phenotype similar to the double mutant of DNA topoisomerases I and II but contains normal levels of both DNA topoisomerase activities.

Journal Article↗
PubMed1994

Linker insertion mutagenesis of Drosophila topoisomerase II. Probing the structure of eukaryotic topoisomerase II.

The sequences of all type II DNA topoisomerases, and possibly some of their key structural features, are conserved. The N-terminal and middle regions of the eukaryotic DNA topoisomerase II are homologous to the bacterial gyrase subunits B and A, respectively, and the hydrophilic C-terminal region is more divergent among these enzymes. To gain further insights into the structure of eukaryotic topoisomerase II, we constructed 23 linker insertion mutants of Drosophila DNA topoisomerase II. These mutant proteins were expressed in a heterologous yeast system, in which we have previously demonstrated that Drosophila DNA topoisomerase II could be functionally expressed and complement yeast top2 mutations. The linker insertion mutants were characterized genetically by testing for complementation of yeast top2ts mutation at the non-permissive temperature and complementation of yeast top2 null mutation using a color sector assay. We also partially purified the mutant proteins and examined their enzymatic activity by unknotting the P4 knotted DNA. There appears to be a good correlation between the in vivo and in vitro activities. There are nine fully active, six partially active, and eight negative linker insertion mutants. All five linker insertion mutants in the C-terminal region are active and two linker insertion mutants located in the junction of the two regions homologous to gyrB/gyrA subunits are also active. In addition, we also mapped the trypsin-sensitive sites in Drosophila DNA topoisomerase II. The C-terminal region is extremely sensitive to trypsin digestion. Another major trypsin-sensitive site is located between Lys406 and Thr407, which is near the protease sites also observed in the bacterial gyrB subunit and yeast topoisomerase II. We discuss the possible structural and functional implications of these results.

Amino Acid Sequence↗
PubMed1995

A large circular minichromosome of Schizosaccharomyces pombe requires a high dose of type II DNA topoisomerase for its stabilization.

We have constructed circular minichromosomes, ranging in size from 36 to 110 kb, containing the centromeric repeats of Schizosaccharomyces pombe cen3. Comparison of their mitotic stability showed that the circular minichromosomes became more unstable with increasing in size, however, a linear cen3 minichromosome, which is almost the same size as the largest circular one tested, does not show such instability. High levels of expression of the top2+ (type II DNA topoisomerase; topo II) but not top1+ gene (type I DNA topoisomerase) suppressed the instability of the largest circular minichromosome, whereas partial inactivation of topo II dramatically destabilized the minichromosome. A mutant topo II, defective in nuclear localization but still retaining its in vitro relaxation activity, did not stabilize the circular minichromosome. These results indicate that endogenous type II DNA topoisomerase is insufficient for accurate segregation of the circular minichromosome. In addition, the replication of the minichromosomal DNA appears to proceed normally, because the presence of the unstable minichromosome did not cause G2 delay. A likely cause of the instability is intertwining of the minichromosome DNA possibly occurring after DNA replication. An interaction between topo II and the centromeric repeats is implied by the finding that multiple copies of the centromeric repeat, dg-dh, affect stability of the minichromosome similarly to top2+ gene dosage.

Chromosomes, Fungal↗
PubMed1988

Deficiency in both type I and type II DNA topoisomerase activities differentially affect rRNA and ribosomal protein synthesis in Schizosaccharomyces pombe.

The synthesis of rRNA and r-proteins was studied in temperature-sensitive topoisomerase mutants of the fisson yeast Schizosaccharomyces pombe. To reduce the severity of heatshock response seen in the wild type strain, slow temperature shift-up of the cultures was used to inactivate the mutant topoisomerases. It was found that the temperature shift caused a large preferential reduction of rRNA synthesis in the top1top2 double mutant. In contrast, no preferential inhibition of rRNA synthesis was observed in top1 or top2 single mutants, although some reduction in the total RNA synthesis was observed in the top2 mutant. Thus, as observed with Saccharomyces cerevisiae (Brill et al. 1987), relaxation of supercoiled DNA structures by either topoisomerase I or II appears to be essential for efficient transcription of rRNA genes. Analysis of r-protein synthesis indicated that there were small decreases in the differential synthesis rates of r-proteins after temperature shift-up in the top1top2 mutant, but the observed negative effects on r-protein synthesis was much smaller than that on rRNA synthesis, and degradation of the newly synthesized r-proteins was observed. These observations indicate the apparent lack of tight coupling between rRNA and r-protein synthesis in S. pombe under these experimental conditions.

DNA Topoisomerases, Type I↗
PubMed1999

Carbamate analogues of amsacrine active against non-cycling cells: relative activity against topoisomerases IIalpha and beta.

PURPOSE: Methyl N-(4'-(9-acridinylamino)-phenyl)carbamate hydrochloride (AMCA) and methyl N-(4'-(9-acridinylamino)-2-methoxyphenyl)carbamate hydrochloride (mAMCA) are analogues of the topoisomerase II (topo II) poison amsacrine, and are distinguished from amsacrine by their high cytotoxicity towards non-cycling cells. Since mammalian cells contain two forms (alpha and beta) of topo II and the alpha isoform is down-regulated in non-cycling cells, we have considered whether these carbamate analogues target topo IIbeta selectively. METHODS: A drug permeable yeast strain (JN394 top2-4) was transformed using a shuttle vector containing either human top2alpha, human top2alpha or yeast top2 under the control of a GAL1 promoter. The strain was analysed at a non-permissive temperature, where only the plasmid-borne topo II was active. RESULTS: AMCA and mAMCA produced comparable levels of cell killing with human DNA topo IIalpha, human DNA topo IIbeta and yeast DNA topo II. Two other acridine derivatives N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and its 7-chloro derivative, which like AMCA and mAMCA are able to overcome multidrug resistance mechanisms, were much more active against human DNA topo IIalpha than against human DNA topo IIbeta and yeast DNA topo II. A series of mutant Chinese hamster and human lines with defined topo lesions, including the HL60/MX2 line that lacks topo IIbeta expression, was also used to compare resistance to amsacrine, AMCA and etoposide. Loss of topo IIbeta activity had a greater effect on amsacrine and AMCA than on etoposide. Resistance of murine Lewis lung cultures in exponential and plateau phase was also measured. Loss of topo IIalpha activity, as measured in both mutant cells expressing lower amounts of enzyme and in cells in plateau phase, resulted in concomitant acquisition of resistance that was greatest for etoposide and least for AMCA. CONCLUSION: We conclude that the carbamate analogues of amsacrine recognize both topo IIalpha and beta in cells.

Acridines↗
PubMed1999

The topoisomerase II-associated protein, Pat1p, is required for maintenance of rDNA locus stability in Saccharomyces cerevisiae.

The Pat1 protein of Saccharomyces cerevisiae was identified during a screen for proteins that interact with topoisomerase II. Previously, we have shown that pat1 delta mutants exhibit a slow-growth phenotype and an elevated frequency of both mitotic and meiotic chromosome mis-segregation. Here, we have studied the effects of deleting the PAT1 gene on chromosomal stability, with particular reference to rates of homologous recombination within the rDNA locus. This locus was analyzed because rDNA-specific hyperrecombination is known to occur in conditional top2 mutants. We show that pat1 delta strains mimic top2 mutants in displaying an elevated rate of intrachromosomal excision recombination at the rDNA locus, but not elsewhere in the genome. The elevated rate of recombination is dependent upon Rad52p, but not upon Rad51p or Rad54p. However, pat1 delta strains display additional manifestations of more general genomic instability, in that they show mild sensitivity to UV light and an increased incidence of interchromosomal recombination between heteroalleles.

Alleles↗
PubMed1992

Two independent amsacrine-resistant human myeloid leukemia cell lines share an identical point mutation in the 170 kDa form of human topoisomerase II.

Cloning and sequencing of cDNA segments of human TOP2 gene encoding the 170 kDa form of human DNA topoisomerase II show that Arg486 of the enzyme has been mutated to a lysine in the enzyme from two human leukemia cell lines HL-60/AMSA and KBM-3/AMSA, which were independently selected for resistance to the antitumor drug amsacrine (4'-[9-acridinylamino]-methanesulfon-m-anisidide, mAMSA). Sequence identity comparisons between eukaryotic DNA topoisomerase II and bacterial gyrase (bacterial DNA topoisomerase II) indicate that the position of the common mutation observed in mAMSA-resistant human TOP2 corresponds to that of the point mutation nal-31 in the Escherichia coli gyrase B gene, which confers resistance to nalidixic acid. Because mAMSA and nalidixic acid are known to act on their respective targets by a common mechanism of trapping the covalent enzyme-DNA intermediates, these results provide strong evidence that the 170 kDa form of human DNA topoisomerase II is a major cellular target of mAMSA, and that Arg486 of this enzyme is involved in mAMSA-mediated trapping of the covalent enzyme-DNA complex.

Amino Acid Sequence↗
PubMed1989

A subthreshold level of DNA topoisomerases leads to the excision of yeast rDNA as extrachromosomal rings.

In a yeast DNA topoisomerase double mutant TG205 (delta top1 top2-4), over half of the rDNA is present as extrachromosomal rings containing one 9 kb unit of the rDNA gene or tandem repeats of it. Expression of a plasmid-borne TOP1 or TOP2 gene in the strain leads to the integation of the extrachromosomal rDNA rings back into the chromosomal rDNA cluster. When the plasmid-borne topoisomerase gene is expressed from an inducible promoter of the GAL1 gene, repression of the gene by dextrose leads to reappearance of the extrachromosomal rDNA rings. The DNA topoisomerase-dependent excision/integration of rDNA is discussed in terms of the possibility of rDNA supercoiling by transcription and the effects of DNA topology on intra- and interchromosomal recombination.

DNA Topoisomerases, Type I↗
PubMed1999

Synthesis of cytotoxic indenoisoquinoline topoisomerase I poisons.

A number of indenoisoquinolines were prepared and evaluated for cytotoxicity in human cancer cell cultures and for activity vs topoisomerase 1 (top1). The two most cytotoxic indenoisoquinolines proved to be cis-6-ethyl-5,6,12,13-tetrahydro-2,3-dimethoxy-8, 9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (21) and cis-6-allyl-5,6,12,13-tetrahydro-2,3-dimethoxy-8, 9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (22), both of which displayed submicromolar mean graph midpoints when tested in 55 human cancer cell cultures. Two of the most potent top1 inhibitors were 6-(3-carboxy-1-propyl)-5,6-dihydro-5, 11-dioxo-11H-indeno[1,2-c]isoquinoline (26) and 6-ethyl-2, 3-dimethoxy-8,9-(methylenedioxy)-11H-indeno[1,2-c]isoquinolinium chloride (27), both of which also inhibited top2, unwound DNA, and are assumed to be DNA intercalators. However, two additional potent top1 inhibitors, 6-allyl-5,6-dihydro-2,3-dimethoxy-8, 9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (13c) and 5,6-dihydro-6-(4-hydroxybut-1-yl)-2,3-dimethoxy-8, 9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (19a), did not unwind DNA and did not affect top2. Some of the DNA cleavage sites detected in the presence of the indenoisoquinolines were different from those seen with the camptothecins. The cleavage sites induced by the indenoisoquinolines were reversed by salt treatment, which is consistent with the reversible trapping of top1 cleavable complexes by the indenoisoquinolines. In general, the potencies of the indenoisoquinolines as top1 inhibitors did not correlate with their potencies as cytotoxic agents, as some of the most cytotoxic agents had little if any effect on top1. On the other hand, the most potent of the indenoisoquinolines vs top1 were not the most cytotoxic. In several cases, moderate activity was observed for both cytotoxicity and activity vs top1.

Antineoplastic Agents↗
PubMed2002

A GyrB-GyrA fusion protein expressed in yeast cells is able to remove DNA supercoils but cannot substitute eukaryotic topoisomerase II.

BACKGROUND: Type II topoisomerases are a highly conserved class of enzymes which transport one double-stranded DNA segment through a transient break in another. Whereas the eukaryotic enzymes are homodimers of a single polypeptide, their bacterial homologues are homodimers of two independently coded protein subunits. Unlike eukaryotic topoisomerase II and bacterial topoisomerase IV, DNA gyrase is a bacterial type II topoisomerase which specializes in intramolecular DNA transport. RESULTS: We have fused the Escherichia coli coding sequences for the proteins GyrB and GyrA, which comprise DNA gyrase. This fusion was expressed in yeast cells and yielded the expected full-length protein product. When it was expressed in Deltatop1- top2-4 yeast cells, the fusion protein compensated their slow growth and reverted their elevated chromosomal excision of ribosomal genes. Furthermore, it removed DNA positive supercoils. The fusion protein, however, was unable to complement the temperature-dependent lethality of top2-4 cells. CONCLUSION: Fusion of the E. coli GyrB and GyrA proteins leads to a catalytically active topoisomerase which compensates several phenotypic traits attributed to unconstrained DNA supercoiling in topoisomerase-deficient cells. However, since the fusion protein cannot substitute for topoisomerase II, it may be efficient in intramolecular but not intermolecular DNA passage, resembling the catalytic properties of DNA gyrase.

DNA Gyrase↗
PubMed2000

Assignment of functional amino acids around the active site of human DNA topoisomerase IIalpha.

An expression library for active site mutants of human topoisomerase IIalpha (TOP2alpha) was constructed by replacing the sequence encoding residues 793-808 with a randomized oligonucleotide cassette. This plasmid library was transformed into a temperature-sensitive yeast strain (top2-1), and viable transformants were selected at the restrictive temperature. Among the active TOP2alpha mutants, no substitution was allowed at Tyr(805), the 5' anchor of the cleaved DNA, and only conservative substitutions were allowed at Leu(794), Asp(797), Ala(801), and Arg(804). Thus, these 5 residues are critical for human TOP2alpha activity, and the remaining mutagenized residues are less critical for function. Using the x-ray crystal structure of yeast TOP2 as a structural model, it can be deduced that these 5 functionally important residues lie in a plane. One of the possible functions of this plane may be that it interacts with the DNA substrate upon catalysis. The side chains of Ser(803) and Lys(798), which confer drug resistance, lie adjacent to this plane.

Amino Acid Sequence↗
PubMed2005

Stability of the topoisomerase II closed clamp conformation may influence DNA-stimulated ATP hydrolysis.

Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II alpha with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr28 --> Phe) was introduced in yeast Top2. The ATPase activities of human TOP2alpha(Tyr50 --> Phe) and yeast Top2(Tyr28 --> Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.

Adenosine Diphosphate↗