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Etoposide is an important chemotherapeutic agent that is used to treat a wide spectrum of human cancers. It has been in clinical use for more than two decades and remains one of the most highly prescribed anticancer drugs in the world. The primary cytotoxic target for etoposide is topoisomerase II. This ubiquitous enzyme regulates DNA under- and overwinding, and removes knots and tangles from the genome by generating transient double-stranded breaks in the double helix. Etoposide kills cells by stabilizing a covalent enzyme-cleaved DNA complex (known as the cleavage complex) that is a transient intermediate in the catalytic cycle of topoisomerase II. The accumulation of cleavage complexes in treated cells leads to the generation of permanent DNA strand breaks, which trigger recombination/repair pathways, mutagenesis, and chromosomal translocations. If these breaks overwhelm the cell, they can initiate death pathways. Thus, etoposide converts topoisomerase II from an essential enzyme to a potent cellular toxin that fragments the genome. Although the topoisomerase II-DNA cleavage complex is an important target for cancer chemotherapy, there also is evidence that topoisomerase II-mediated DNA strand breaks induced by etoposide and other agents can trigger chromosomal translocations that lead to specific types of leukemia. Given the central role of topoisomerase II in both the cure and initiation of human cancers, it is imperative to further understand the mechanism by which the enzyme cleaves and rejoins the double helix and the process by which etoposide and other anticancer drugs alter topoisomerase II function.

Although benzene induces leukemias in humans, the compound is not believed to generate chromosomal damage directly. Rather, benzene is thought to act through a series of phenolic- and quinone-based metabolites, especially 1,4-benzoquinone. A recent study found that 1,4-benzoquinone is a potent topoisomerase II poison in vitro and in cultured human cells [Lindsey et al. (2004) Biochemistry 43, 7363−7374]. Because benzene is metabolized to multiple compounds in addition to 1,4-benzoquinone, we determined the effects of several phenolic metabolites, including catechol, 1,2,4-benzenetriol, 1,4-hydroquinone, 2,2‘-biphenol, and 4,4‘-biphenol, on the DNA cleavage activity of human topoisomerase IIα. Only 1,4-hydroquinone generated substantial levels of topoisomerase II-mediated DNA scission. DNA cleavage with this compound approached levels observed with 1,4-benzoquinone (~5- vs 8-fold) but required a considerably higher concentration (~250 vs 25 μM). 1,4-Hydroquinone is a precursor to 1,4-benzoquinone in the body and can be activated to the quinone by redox cycling. It is not known whether the effects of 1,4-hydroquinone on human topoisomerase IIα reflect a lower reactivity of the hydroquinone or a low level of activation to the quinone. The high concentration of 1,4-hydroquinone required to increase enzyme-mediated DNA cleavage is consistent with either explanation. 1,4-Hydroquinone displayed attributes against topoisomerase IIα, including DNA cleavage specificity, that were similar to those of 1,4-benzoquinone. However, 1,4-hydroquinone consistently inhibited DNA ligation to a greater extent than 1,4-benzoquinone. This latter result implies that the hydroquinone may display (at least in part) independent activity against topoisomerase IIα. The present findings are consistent with the hypothesis that topoisomerase IIα plays a role in the initiation of specific types of leukemia that are induced by benzene and its metabolites.

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIalpha and beta were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N(6)-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage approximately 10-fold. 1,N(6)-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.

The DNA strand passage activity of eukaryotic topoisomerase II relies on a cascade of conformational changes triggered by ATP binding to the N-terminal domain of the enzyme. To investigate the interdomain communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha, we characterized a mutant enzyme that contains a deletion at the interface between the two domains, covering amino acids 350-407. The ATPase domain retained full activity with a rate of ATP hydrolysis that was severalfold higher than normal, but the ATPase activity was unaffected by DNA. The cleavage and religation activities of the enzyme were comparable with those of the wild-type enzyme both in the absence and presence of cancer chemotherapeutic agents. However, neither ATP nor a nonhydrolyzable ATP analog stimulated cleavage complex formation. Although both conserved domains retained full activity, the mutant enzyme was unable to coordinate these activities into strand passage. Our findings suggest that the normal conformational transitions occurring in the enzyme upon ATP binding are hampered or lacking in the mutant enzyme. Consistent with this hypothesis, the enzyme displayed an abnormal clamp closing activity. In summary, the region covering amino acids 350-407 in human topoisomerase IIalpha seems to be essential for correct interdomain communication and probably is involved in signaling ATP binding to the rest of the enzyme.

Type II topoisomerases, a family of enzymes that govern topological DNA interconversions, are essential to many cellular processes in eukaryotic organisms. Because no data are available about the functions of these enzymes in the replication of viruses that infect eukaryotic hosts, this led us to express and characterize the first topoisomerase II encoded by one of such viruses. Paramecium bursaria chlorella virus 1 (PBCV-1) infects certain chlorella-like green algae and encodes a 120-kDa protein with a similarity to type II topoisomerases. This protein was expressed in Saccharomyces cerevisiae and was highly active in relaxation of both negatively and positively supercoiled plasmid DNA, catenation of plasmid DNA, and decatenation of kinetoplast DNA networks. Its optimal activity was determined, and the omission of Mg(2+) or its replacement with other divalent cations abolished DNA relaxation. All activities of the recombinant enzyme were ATP dependent. Increasing salt concentrations shifted DNA relaxation from a normally processive mechanism to a distributive mode. Thus, even though the PBCV-1 enzyme is considerably smaller than other eukaryotic topoisomerase II enzymes (whose molecular masses are typically 160-180 kDa), it displays all the catalytic properties expected for a type II topoisomerase.

Bisdioxopiperazines are a unique class of topoisomerase II inhibitors that lock topoisomerase II at a point in the enzyme reaction cycle where the enzyme forms a closed clamp around DNA. We examined cell killing by ICRF-187 and ICRF-193 in yeast cells expressing human topoisomerase II alpha (htop-IIalpha). Expression of htop-IIalpha in yeast cells sensitizes them to both ICRF-187 and ICRF-193, compared with cells expressing yeast topoisomerase II. ICRF-193 is still able to exert growth inhibition in the presence of genes encoding both ICRF-193-resistant and ICRF-193-sensitive htop-IIalpha enzymes, indicating that sensitivity to bisdioxopiperazines is dominant. Killing by ICRF-193 occurs more rapidly, than the killing in yeast cells due to a temperature-sensitive yeast topoisomerase II incubated at the non-permissive temperature. These results are reminiscent of a top-II poison such as etoposide. However, the killing caused by ICRF-193 and ICRF-187 is not enhanced by mutations in the RAD52 pathway. The levels of drug-induced DNA cleavage observed with htop-IIalpha in vitro is insufficient to explain the sensitivity induced by this enzyme in yeast cells. Finally, arrest of cells in G(1) does not protect cells from ICRF-193 lethality, a result inconsistent with killing mechanisms due to catalytic inhibition of top-II or stabilization of a cleavable complex. We suggest that the observed pattern of cell killing is most consistent with a poisoning of htop-II by ICRF-193 by a novel mechanism. The accumulation of closed clamp conformations of htop-II induced by ICRF-193 that are trapped on DNA might interfere with transcription, or other DNA metabolic processes, resulting in cell death.

Topoisomerase II is an essential enzyme that is the target for several clinically important anticancer drugs. Although this enzyme must create transient double-stranded breaks in the genetic material in order to carry out its indispensable DNA strand passage reaction, the factors that underlie its nucleotide cleavage specificity remain an enigma. Therefore, to address the critical issue of enzyme specificity, a modified systematic evolution of ligands by exponential enrichment (SELEX) protocol was employed to select/evolve DNA sequences that were preferentially cleaved by Drosophila melanogaster topoisomerase II. Levels of DNA scission rose substantially (from 3 to 20%) over 20 rounds of SELEX. In vitro selection/evolution converged on an alternating purine/pyrmidine sequence that was highly AT-rich (TATATATACATATATATA). The preference for this sequence was more pronounced for Drosophila topoisomerase II over other species and was increased in the presence of DNA cleavage-enhancing anticancer drugs. Enhanced cleavage appeared to be based on higher rates of DNA scission rather than increased binding affinity or decreased religation rates. The preferred sequence for topoisomerase II-mediated DNA cleavage is dramatically overrepresented ( approximately 10,000-fold) in the euchromatic genome of D. melanogaster, implying that it may be a site for the physiological action of this enzyme.

Quinolones are the most active oral antibacterials in clinical use and act by increasing DNA cleavage mediated by prokaryotic type II topoisomerases. Although topoisomerase IV appears to be the primary cytotoxic target for most quinolones in Gram-positive bacteria, interactions between the enzyme and these drugs are poorly understood. Therefore, the effects of ciprofloxacin on the DNA cleavage and religation reactions of Staphylococcus aureus topoisomerase IV were characterized. Ciprofloxacin doubled DNA scission at 150 nM drug and increased cleavage approximately 9-fold at 5 microM. Furthermore, it dramatically inhibited rates of DNA religation mediated by S. aureus topoisomerase IV. This inhibition of religation is in marked contrast to the effects of antineoplastic quinolones on eukaryotic topoisomerase II, and suggests that the mechanistic basis for quinolone action against type II topoisomerases has not been maintained across evolutionary boundaries. The apparent change in quinolone mechanism was not caused by an overt difference in the drug interaction domain on topoisomerase IV. Therefore, we propose that the mechanistic basis for quinolone action is regulated by subtle changes in drug orientation within the enzyme.drug.DNA ternary complex rather than gross differences in the site of drug binding.

Cytosine arabinoside (araC) is an important drug used for the treatment of human leukemias. In order to exert its cytotoxic effects, araC must be incorporated into chromosomal DNA. Although specific DNA lesions that involve base loss or modification stimulate nucleic acid cleavage mediated by type II topoisomerases, the effects of deoxyribose sugar ring modification on enzyme activity have not been examined. Therefore, the effects of incorporated araC residues on the DNA cleavage/religation equilibrium of human topoisomerase IIalpha and beta were characterized. AraC lesions were position-specific topoisomerase II poisons and stimulated DNA scission mediated by both human type II enzymes. However, the positional specificity of araC residues differed from that previously reported for other cleavage-enhancing DNA lesions. Finally, additive or synergistic increases in DNA cleavage were observed in the presence of araC lesions and etoposide. These findings broaden the range of DNA lesions known to alter topoisomerase II function and raise the possibility that this enzyme may mediate some of the cellular effects of araC.

To further characterize the mechanistic basis for cellular resistance/hypersensitivity to anticancer drugs, a yeast genetic system was used to select a mutant type II topoisomerase that conferred cellular resistance to CP-115,953, amsacrine, etoposide, and ellipticine. The mutant enzyme contained a single point mutation that converted Gly437 --> Ser (top2G437S). Purified top2G437S displayed wild-type enzymatic activity in the absence of drugs but exhibited two properties that were not predicted by the cellular resistance phenotype. First, in the absence of ATP, it was hypersensitive to all of the drugs examined and hypersensitivity correlated with increased drug affinity. Second, in the presence of ATP, top2G437S lost its hypersensitivity and displayed wild-type drug sensitivity. Since the resistance of yeast harboring top2G437S could not be explained by alterations in enzyme-drug interactions, physiological levels of topoisomerase II were determined. The Gly437 --> Ser mutation reduced the stability of topoisomerase II and decreased the cellular concentration of the enzyme. These findings suggest that the physiological drug resistance phenotype conferred by top2G437S results primarily from its decreased stability. This study highlights the need to analyze both the biochemistry and the physiology of topoisomerase II mutants with altered drug sensitivity in order to define the mechanistic bridge that links enzyme function to cellular phenotype.

Eukaryotic topoisomerases recognize DNA topology and preferentially react with positively or negatively supercoiled molecules over relaxed substrates. To elucidate the mechanism of this recognition, we examined the interaction of topoisomerases with DNA by electron microscopy. Under all conditions employed, approximately 90% of the bound type I or II enzyme was observed at points of helix–helix juxtaposition on negatively supercoiled plasmids which contained as few as four crossovers. Recognition was independent of torsional stress, as enzyme molecules were also found at crossovers on linear DNA. Since juxtaposed helices are more prevalent in supercoiled compared with relaxed nucleic acids, we propose that eukaryotic topoisomerases I and II recognize underwound or overwound substrates by interacting preferentially with DNA crossovers. This may represent a general mechanism for the recognition of DNA topology by proteins.

Several clinically relevant anticancer drugs induce genomic mutations and cell death by increasing topoisomerase II-mediated DNA breakage. To determine whether endogenous DNA damage also affects this cleavage event, the effects of abasic sites (the most commonly formed spontaneous DNA lesion) on topoisomerase II activity were investigated. The presence of 3 abasic sites/plasmid stimulated enzyme-mediated DNA breakage > 6-fold, primarily by enhancing the forward rate of cleavage. This corresponds to a potency that is > 2000-fold higher than that of the anticancer drug, etoposide. These findings suggest that abasic sites represent endogenous topoisomerase II poisons and imply that anticancer drugs mimic the cleavage-enhancing actions of naturally occurring DNA lesions.

In prokaryotic type II topoisomerases (DNA gyrases), mutations that result in resistance to quinolones frequently occur at Ser83 or Ser84 of the gyrA subunit. Mutations to Trp, Ala, and Leu have been identified, all of which confer high levels of quinolone resistance. Extensive segments of DNA gyrase are homologous to eukaryotic topoisomerase II, and Ser741 of yeast TOP2 is homologous to Ser83 of prokaryotic DNA gyrA. Introduction of the Ser741-->Trp mutation into yeast TOP2 confers resistance to 6,8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl- 4-quinolone-3-carboxylic acid (CP-115,953), a fluoroquinolone with substantial activity against eukaryotic topoisomerase II, whereas changing Ser741 to either Leu or Ala does not change sensitivity to quinolones. Interestingly, Ser741-->Trp in the yeast TOP2 also confers hypersensitivity to etoposide. Sensitivity to intercalating anti-topoisomerase II agents such as amsacrine is not changed by any of the three mutations. The topoisomerase II protein carrying the Ser741-->Trp mutation was overexpressed and purified. The purified mutant enzyme had enhanced levels of etoposide stabilized covalent complex as compared with the wild type enzyme and reduced cleavage with CP-115,953. Unlike the wild type enzyme, etoposide-stabilized cleavage is not readily reversible by heat. We suggest that Ser741 is near a binding site for both quinolones and etoposide and that the Ser741-->Trp mutation leads to a more stable ternary complex between etoposide, DNA, and the mutant enzyme.

The carbonate binding site on horse cytochrome c was mapped by comparing the yields of carboxydinitrophenyl-cytochromes c, each with a single carboxydinitrophenyl-substituted lysine residue per molecule, when the modification reaction was carried out in the presence and absence of carbonate. The site is located on the "left surface" of the protein and consists of lysine residues 72 and/or 73 as well as 86 and/or 87 (Carbonate Site). Although one of the binding sites for phosphate on cytochrome c (Phosphat Site I) is located near the carbonate site, the sites are distinctly different since carbonate does not displace bound phosphate, as monitored by 31P NMR. Furthermore, citrate interacts with Phosphate Site I with high affinity, whereas chloride, acetate, borate, and cacodylate have a much lower affinity for this site, if they bind to it at all. The affinity of phosphate for Phosphate Site I (KD = 2 X 10(-4) M) is at least 1 order of magnitude higher than it is for other sites of interaction.

A previous study (M.J. Robinson, B.A. Martin, T.D. Gootz, P.R. McGuirk, M. Moynihan, J.A. Sutcliffe, and N. Osheroff, J. Biol. Chem. 266:14585-14592, 1991) demonstrated that novel 6,8-difluoroquinolones were potent effectors of eukaryotic topoisomerase II. To determine the contribution of the C-8 fluorine to drug potency, we compared the effects of CP-115,955 [6-fluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid] on the enzymatic activities of Drosophila melanogaster topoisomerase II with those of CP-115,953 (the 6,8-difluoro parent compound of CP-115,955). Removal of the C-8 fluoro group decreased the ability of the quinolone to enhance enzyme-mediated DNA cleavage approximately 2.5-fold. Like its difluorinated counterpart, CP-115,955 increased the levels of cleavage intermediates without impairing the DNA religation reaction of the enzyme. Removal of the C-8 fluorine reduced the ability of the quinolone to inhibit topoisomerase II-catalyzed DNA relaxation. In addition, the cytotoxicity of CP-115,955 towards Chinese hamster ovary cells was decreased compared with that of CP-115,953. These results demonstrate that the C-8 fluorine increases the potency of quinolone derivatives against eukaryotic topoisomerase II and mammalian cells. Further comparisons of CP-115,955 with CP-115,953 and CP-67,804 (the N-1 ethyl-substituted derivative of the difluoro parent compound) indicate that the two intrinsic activities of quinolone-based drugs towards topoisomerase II (i.e., enhancement of DNA cleavage and inhibition of catalytic strand passage) can be differentially influenced by alteration of ring substituents. Finally, correlations between the biochemical and cytological activities of these drugs suggest that the ability to inhibit catalytic strand passage enhances the cytotoxic potential of quinolones towards eukaryotic cells.

In order to characterize more fully the mechanism by which casein kinase II is regulated in mammalian cells, the effect of epidermal growth factor (EGF) on the activity of the kinase in human A-431 carcinoma cells was examined. Treatment of cells with EGF prior to lysis consistently resulted in a transient 4-fold increase in the activity of cytosolic casein kinase II. Activity rose sharply between 20 and 30 min, peaked at approximately 50 min, and returned to basal levels by approximately 120 min. Similar results were obtained using the casein kinase II specific peptide substrate, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu, or DNA topoisomerase II (which is specifically modified by the kinase in vivoand serves as a high affinity substrate in vitro) as the phosphate acceptor in assays. Identification of casein kinase II as the stimulated activity was confirmed by partial proteolytic mapping and phosphoamino acid analysis of modified topoisomerase II, by inhibition at nanomolar levels of heparin or micromolar levels of nonradioactive GTP, and by the ability to employ radioactive GTP as a direct phosphate donor. The EGF stimulation of casein kinase II was dependent on the availability of intracellular (but not extracellular) calcium. In addition, hormonal action was modulated by calcium/phospholipid-dependent protein kinase (protein kinase C). Casein kinase II stimulation did not require an increase in the concentration of the kinase, protein synthesis, the continual presence of a small effector molecule, or a direct interaction with the EGF receptor/tyrosine kinase. In contrast, hormonal activation of the kinase was dependent on the phosphorylation of casein kinase II or a terminal stimulatory factor.

The interaction of topoisomerase II with its DNA cleavage site is critical to the physiological functions of the enzyme. Despite this importance, the specific enzyme-DNA interactions that drive topoisomerase II-mediated DNA cleavage and religation are poorly understood. Therefore, to dissect interactions between the enzyme and its cleavage site, abasic DNA lesions were incorporated into a bilaterally symmetrical and identical cleavage site. Results indicate that topoisomerase II has unique interactions with each position of the 4-base overhang generated by enzyme-mediated DNA cleavage. Lesions located 2 bases 3' to the point of scission stimulated cleavage the most, whereas those 3 bases from the point of scission stimulated cleavage the least. Moreover, an additive and in some cases synergistic cleavage enhancement was observed in oligonucleotides that contained multiple DNA lesions, with levels reaching >60-fold higher than the wild-type substrate. Finally, topoisomerase II efficiently cleaved and religated a DNA substrate in which apyrimidinic sites were simultaneously incorporated at every position on one strand of the 4-base overhang. Therefore, unlike classical DNA ligases in which base pairing is the driving force behind closure of the DNA break, it appears that for topoisomerase II, the enzyme is responsible for the spatial orientation of the DNA termini for ligation.

Topoisomerase IV is a bacterial type II topoisomerase that is essential for proper chromosome segregation and is a target for quinolone-based antimicrobial agents. Despite the importance of this enzyme to the survival of prokaryotic cells and to the treatment of bacterial infections, relatively little is known about the details of its catalytic mechanism or the basis by which quinolones alter its enzymatic functions. Therefore, a series of experiments that analyzed individual steps of the topoisomerase IV catalytic cycle were undertaken to address these critical mechanistic issues. The following conclusions were drawn. First, equilibrium levels of DNA cleavage mediated by the bacterial enzyme were considerably (>10-fold) higher than those observed with its eukaryotic counterparts. To a large extent, this reflected decreased rates of DNA religation. Second, the preference of topoisomerase IV for catalyzing DNA decatenation over relaxation reflects increased rates of strand passage and enzyme recycling rather than a heightened recognition of intermolecular DNA helices. Third, quinolones stimulate topoisomerase IV-mediated DNA cleavage both by increasing rates of DNA scission and by inhibiting religation of cleaved DNA. Finally, quinolones inhibit the overall catalytic activity of topoisomerase IV primarily by interfering with enzyme-ATP interactions.

Merbarone is a catalytic inhibitor of topoisomerase II that is in clinical trials as an anticancer agent. Despite the potential therapeutic value of this drug, the mechanism by which it blocks topoisomerase II activity has not been delineated. Therefore, to determine the mechanistic basis for the inhibitory action of merbarone, the effects of this drug on individual steps of the catalytic cycle of human topoisomerase IIalpha were assessed. Concentrations of merbarone that inhibited catalytic activity >/=80% had no effect on either enzyme.DNA binding or ATP hydrolysis. In contrast, the drug was a potent inhibitor of enzyme-mediated DNA scission (in the absence or presence of ATP), and the inhibitory profiles of merbarone for DNA cleavage and relaxation were similar. These data indicate that merbarone acts primarily by blocking topoisomerase II-mediated DNA cleavage. Merbarone inhibited DNA scission in a global (rather than site-specific) fashion but did not appear to intercalate into DNA or bind in the minor groove. Since the drug competed with etoposide (a cleavage-enhancing agent that binds directly to topoisomerase II), it is proposed that merbarone exerts its inhibitory effects through interactions with the enzyme and that the drug shares an interaction domain on topoisomerase II with cleavage-enhancing agents.

The phosphorylation of DNA topoisomerase II in Drosophila Kc tissue culture cells was characterized by in vivo labeling studies and in vitro studies that examined the modification of exogenous enzyme in total homogenates of these embryonic cells. Several lines of evidence identified casein kinase II as the kinase primarily responsible for phosphorylating DNA topoisomerase II. First, the only amino acyl residue modified in the enzyme was serine. Second, partial proteolytic maps of topoisomerase II which had been labeled with [32P]phosphate by Drosophila cells in vivo, by cell homogenates in vitro, or by purified casein kinase II were indistinguishable from one another. Third, phosphorylation in cell homogenates was inhibited by micrograms/ml concentrations of heparin, micromolar concentrations of nonradioactive GTP, or anti-Drosophila casein kinase II antiserum. Fourth, cell homogenates were able to employ [gamma-32P]GTP as a phosphate donor nearly as well as [gamma-32P]ATP. Although topoisomerase II was phosphorylated in homogenates under conditions that specifically stimulate protein kinase C, calcium/calmodulin-dependent protein kinase, or cAMP-dependent protein kinase, modification was always sensitive to anti-casein kinase II antiserum or heparin. Thus, under a variety of conditions, topoisomerase II appears to be phosphorylated primarily by casein kinase II in the Drosophila embryonic Kc cell system.

Eukaryotic topoisomerase II is capable of binding two separate nucleic acid helices prior to its DNA cleavage and strand passage events (Zechiedrich, E. L., and Osheroff, N (1990) EMBO J. 9, 4555-4562). Presumably, one of these helices represents the helix that the enzyme cleaves (i.e. cleavage helix), and the other represents the helix that it passes (i.e. passage helix) through the break in the nucleic acid backbone. To determine whether the passage helix is required for reaction steps that precede the enzyme's DNA strand passage event, interactions between Drosophila melanogaster topoisomerase II and a short double-stranded oligonucleotide were assessed. These studies employed a 40-mer that contained a specific recognition/cleavage site for the enzyme. The sigmoidal DNA concentration dependence that was observed for cleavage of the 40-mer indicated that topoisomerase II had to interact with more than a single oligonucleotide in order for cleavage to take place. Despite this requirement, results of enzyme DNA binding experiments indicated no binding cooperativity for the 40-mer. These findings strongly suggest a two-site model for topoisomerase II action in which the passage and the cleavage helices bind to the enzyme independently, but the passage helix must be present for efficient topoisomerase II-mediated DNA cleavage to occur.

DNA topoisomerase II has been immunochemically identified on protein blots as a major polypeptide component of the Drosophila nuclear matrix-pore complex-lamina fraction. Indirect immunofluorescence analyses of larval cryosections have confirmed the nuclear localization of topoisomerase II in situ. Although apparently excluded from the nucleolus, the topoisomerase protein is otherwise distributed throughout the interior of interphase nuclei. Similar immunocytochemical studies performed with permeabilized whole giant cells from third-instar larval salivary glands have shown topoisomerase II to be largely restricted to the polytene chromosomes. Upon nuclear disassembly during mitosis, the topoisomerase polypeptide appears to redistribute diffusely throughout the cell. Faint immunofluorescent staining of mitotic chromosomes is also observed.

To determine relationships between the hormonal activation of casein kinase II and its phosphorylation state, epidermal growth factor (EGF)-treated and EGF-naive human A-431 carcinoma cells were cultured in the presence of [32P]orthophosphate. Immunoprecipitation experiments indicated that casein kinase II in the cytosol of EGF-treated cells contained approximately 3-fold more incorporated [32P]phosphate than did its counterpart in untreated cells. Levels of kinase phosphorylation paralleled levels of kinase activity over a wide range of EGF concentrations as well as over a time course of hormone action. Approximately 97% of the incorporated [32P]phosphate was found in the beta subunit of casein kinase II. Both activated and hormone-naive kinase contained radioactive phosphoserine and phosphothreonine but no phosphotyrosine. On the basis of proteolytic mapping experiments, EGF treatment of A-431 cells led to an increase in the average [32P]phosphate content (i.e., hyperphosphorylation) of casein kinase II beta subunit peptides which were modified prior to hormone treatment. Finally, the effect of alkaline phosphatase on the reaction kinetics of activated casein kinase II indicated that hormonal stimulation of the kinase resulted from the increase in its phosphorylation state.

We have purified and characterized an oligopeptide binding protein released from the periplasm of Escherichia coli W by mild osmotic shock. The purified protein was greater than 97% homogeneous as determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 60,000) or isoelectric focusing (pI = 5.95). The binding protein has a Stokes radius of 30 A and a sedimentation coefficient (s(0)20,w) of 4.6 S. Based on these hydrodynamic studies, the native protein has a molecular weight of 56,000. The tripeptide, Ala-Phe-[3H]Gly, which is transported via the shock-sensitive sensitive oligopeptide permease, binds to the purified protein in dilute solution with a Kd of 0.1 microM and a stoichiometry of approximately 1 to 1. Results from this study support the hypothesis that this periplasmic oligopeptide binding protein functions in the initial recognition of peptide substrates for the oligopeptide permease system.

The phosphorylation of Drosophila melanogaster DNA topoisomerase II by purified casein kinase II was characterized in vitro. Under the conditions used, the kinase incorporated a maximum of 2-3 molecules of phosphate per homodimer of topoisomerase II. No autophosphorylation of the topoisomerase was observed. The only amino acid residue modified by casein kinase II was serine. Apparent Km and Vmax values for the phosphorylation reaction were 0.4 microM topoisomerase II and 3.3 mumol of phosphate incorporated per min per mg of kinase, respectively. Phosphorylation stimulated the DNA relaxation activity of topoisomerase II by 3-fold over that of the dephosphorylated enzyme, and the effects of modification could be reversed by treatment with alkaline phosphatase. Therefore, this study demonstrates that post-translational enzymatic modifications can be used to modulate the interaction between topoisomerase II and DNA.

While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover.

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.

A type II DNA topoisomerase has been purified from the nuclei of Drosophila melanogaster 6- to 18-h-old embryos. The enzyme, as assayed by its ability to catenate supercoiled DNA, behaved as a single homogeneous species throughout the procedure and the yield was approximately 0.5 mg of protein/100 g of dechorionated embryos. The final product was entirely ATP-dependent and free of topoisomerase I, endonuclease and protease activities. The purified topoisomerase II had a Stokes radius of 69 A and a sedimentation coefficient (S20,w) of 9.2 S, leading to a calculated native molecular weight of approximately 261,000. The protein consists of a single polypeptide of molecular weight 166,000, as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Taken together with the above hydrodynamic studies, the Drosophila enzyme is probably a homodimer, as has been observed for other eukaryotic type II enzymes. Thus, it appears that during the course of evolution the heterologous subunits which comprise bacterial type II topoisomerases have been combined into a single polypeptide chain in eukaryotes.

Many anticancer drugs "poison" topoisomerase II by enhancing its double-stranded DNA cleavage activity. To determine whether DNA lesions act as endogenous topoisomerase II poisons, we characterized the effects of position-specific apurinic sites on enzyme activity. Lesions located within the 4-base overhang generated by enzyme-mediated DNA scission stimulated cleavage approximately 10-18-fold without altering the specificity of topoisomerase II. DNA breaks were double-stranded in nature, protein-linked, and readily reversible. In contrast, apurinic sites located immediately outside the cleavage overhang were inhibitory. Thus, apurinic sites, which are the most commonly formed lesion in DNA, are position-specific topoisomerase II poisons. A model is proposed that encompasses the actions of endogenous and exogenous topoisomerase II poisons and provides a pre-existing pathway for the cellular actions of topoisomerase II-targeted anticancer drugs.

Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. Cmax) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. Km) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme.drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide. topoisomerase II (rather than etoposide.DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with Cmax values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.

Apurinic sites are position-specific poisons of topoisomerase II and stimulate DNA scission approximately 10-18-fold when they are located within the 4-base overhang generated by enzyme-mediated cleavage (Kingma, P. S., and Osheroff, N. (1997) J. Biol. Chem. 272, 1148-1155). To determine whether other major forms of spontaneous DNA damage also act as topoisomerase II poisons, the effects of position-specific apyrimidinic sites and deaminated cytosines (i.e. uracil:guanine mismatches) on the type II enzyme were determined. Both of these lesions stimulated topoisomerase II-mediated DNA scission with the same positional specificity as apurinic sites but were less efficacious. Moreover, apurinic sites dominated the effects of apyrimidinic sites in substrates that contained multiple lesions. The differential ability of spontaneous lesions to enhance DNA cleavage did not correlate with either a decreased stability of the double helix or the size of the gap formed by base loss. Rather, it appears to be due (at least in part) to increased rates of religation for substrates containing apyrimidinic sites or deaminated cytosines. These results suggest that several forms of spontaneous DNA damage are capable of acting as endogenous poisons of topoisomerase II.

Recently, a number of novel quinolones with potent activity against topoisomerase II and eukaryotic cells have been described. Many of these compounds contain aromatic substituents in their C-7 ring positions. To determine whether pyrimido[1,6-a]benzimidazoles, a class of drugs modeled on quinolones, also display activity toward eukaryotic systems, the effects of Ro 46-7864 and Ro 47-3359 on Drosophila melanogaster topoisomerase II and Kc cells were characterized. While the former drug contains an aliphatic group (4-N-methylpiperazine) at the ring position equivalent to C-7 in quinolones, the latter compound contains an aromatic substituent (2,6-dimethylpyridine). Both pyrimido[1,6-a]benzimidazoles inhibited DNA relaxation catalyzed by the type II enzyme. However, only Ro 47-3359 enhanced topoisomerase II-mediated DNA cleavage and was toxic to Kc cells. At a concentration of 100 microM, this drug approximately doubled the levels of DNA breakage in vitro and killed > 50% of the initial cell population of cultures. These results strongly suggest that selected pyrimido[1,6-a]benzimidazoles may function as topoisomerase II-targeted drugs with cytotoxic potential.

Eukaryotic topoisomerases recognize DNA topology and preferentially react with positively or negatively supercoiled molecules over relaxed substrates. To elucidate the mechanism of this recognition, we examined the interaction of topoisomerases with DNA by electron microscopy. Under all conditions employed, approximately 90% of the bound type I or II enzyme was observed at points of helix–helix juxtaposition on negatively supercoiled plasmids which contained as few as four crossovers. Recognition was independent of torsional stress, as enzyme molecules were also found at crossovers on linear DNA. Since juxtaposed helices are more prevalent in supercoiled compared with relaxed nucleic acids, we propose that eukaryotic topoisomerases I and II recognize underwound or overwound substrates by interacting preferentially with DNA crossovers. This may represent a general mechanism for the recognition of DNA topology by proteins.

Altered sensitivity of topoisomerase II to anticancer drugs profoundly affects the response of eukaryotic cells to these agents. Therefore, several approaches were employed to elucidate the mechanism of drug hypersensitivity of the mutant yeast type II topoisomerase, top2H1012Y. This mutant, which is approximately 5-fold hypersensitive to ellipticine, formed DNA cleavage complexes more rapidly than the wild-type yeast enzyme in the presence of the drug. Conversely, no change in the rate of DNA religation was observed. There was, however, a correlation between increased cleavage rates and enhanced drug binding affinity. The apparent dissociation constant for ellipticine in the mutant topoisomerase II.drug.DNA ternary complex was approximately 5-fold lower than in the wild-type ternary complex. Furthermore, the apparent KD value for the mutant binary (topoisomerase II.drug) complex was approximately 2-fold lower than the corresponding wild-type complex, indicating that drug hypersensitivity is intrinsic to the enzyme. These findings strongly suggest that the enhanced ellipticine binding affinity for topoisomerase II is the mechanistic basis for drug hypersensitivity of top2H1012Y.

A mutant yeast type II topoisomerase was generated by in vitro mutagenesis followed by selection in vivo for resistance to the quinolone CP-115,953. The resulting mutant enzyme had a single point mutation which converted His1012 to Tyr (top2H1012Y). top2H1012Y was overexpressed in yeast, purified, and characterized in vitro. The mutant type II topoisomerase was slightly less active than the wild type enzyme, apparently due to a decreased affinity for DNA. The affinity of the mutant enzyme for ATP was similar to that of wild type topoisomerase II. As determined by DNA cleavage assays, top2H1012Y was resistant to CP-115,953 and etoposide both prior to and following the DNA strand-passage event. In marked contrast, the mutant enzyme displayed wild type sensitivity to amsacrine and was severalfold hypersensitive to ellipticine. A similar pattern of resistance was observed in yeast cells harboring the top2H1012Y allele. Thus, it appears that the mutant type II topoisomerase can distinguish between nonintercalative and intercalative agents. Finally, the His1012-->Tyr mutation defines a potential new drug resistance-conferring region on eukaryotic topoisomerase II.

Although a number of drugs currently in use for the treatment of human cancers act by stimulating topoisomerase II-mediated DNA breakage, little is known regarding interactions between these agents and the enzyme. To further define the mechanism of drug action, interactions between ellipticine (an intercalative drug with clinical relevance) and yeast topoisomerase II were characterized. By utilizing a yeast genetic system, topoisomerase II was identified as the primary cellular target of the drug. Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage. Finally, ellipticine binding to DNA, topoisomerase II, and the enzyme-DNA complex was assessed by steady-state and frequency domain fluorescence spectroscopy. As determined by changes in fluorescence intensity and emission maximum wavelength, and by lifetime analysis, only the protonated species of ellipticine bound to a double-stranded 40-mer oligonucleotide containing a topoisomerase II cleavage site (KD approximately 65 nM). In contrast, predominantly deprotonated ellipticine bound to the enzyme.DNA complex (KD approximately 1.5 microM) or to the enzyme in the absence of nucleic acids (KD approximately 160 nM). These findings suggest that ellipticine interacts directly with topoisomerase II and that the enzyme dictates the ionic state of the drug in the ternary complex. A model is presented in which the topoisomerase II.ellipticine.DNA complex is formed via initial drug binding to either the enzyme or DNA.

CP-115,953 [6,8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4- quinolone-3-carboxylic acid] is a novel quinolone that is highly active against topoisomerase II in vitro and in mammalian cells in culture (M. J. Robinson, B. A. Martin, T. D. Gootz, P. R. McGuirk, M. Moynihan, J. A. Sutcliffe, and N. Osheroff, J. Biol. Chem. 266:14585-14592, 1991). However, the features of the drug that contribute to its activity towards mammalian systems have not been characterized. Therefore, CP-115,953 and a series of related quinolones were examined for their activity against calf thymus topoisomerase II and cultured mammalian cells. CP-115,953 stimulated DNA cleavage mediated by the type II enzyme with a potency that was approximately 600-fold greater than that of the antimicrobial quinolone ciprofloxacin and approximately 50-fold greater than that of the antineoplastic drug etoposide. As determined by the ability to enhance enzyme-mediated DNA cleavage, quinolone activity towards calf thymus topoisomerase II was enhanced by the presence of a cyclopropyl group at the N-1 ring position and by the presence of a fluorine at C-8. Furthermore, the 4'-hydroxyphenyl substituent at the C-7 position was critical for the potency of CP-115,953 towards the mammalian type II enzyme. In this regard, the aromatic nature of the C-7 ring as well as the presence and the position of the 4'-hydroxyl group contributed greatly to drug activity. Finally, the cytotoxicity of quinolones in the CP-115,953 series towards mammalian cells paralleled the in vitro stimulation of DNA cleavage by topoisomerase II rather than the inhibition of enzyme-catalyzed DNA relaxation. This correlation strongly suggests that these quinolones promote cell death by converting topoisomerase II to a cellular poison.

A series of novel C-7 quinolyl-substituted enantiomers of ofloxacin were used to determine the stereospecificity of topoisomerase II for the C-11 methyl group in tricyclic quinolones. In all cases, the S isomer was the most active compound against the eukaryotic enzyme. It was approximately 2.2-fold more potent than the R isomer at inhibiting the overall catalytic activity of topoisomerase II (as monitored by DNA relaxation assays). A markedly greater difference in quinolone activity was observed in enzyme-mediated DNA cleavage reactions. While the S enantiomer stimulated nucleic acid breakage approximately 3.5-fold, the R compound did not enhance and, in fact, decreased initial DNA cleavage levels by approximately 50%. The activity of the racemic mixture more closely resembled that of the R enantiomer. In competition experiments, the DNA cleavage-enhancing effects of the S isomer were attenuated by the R compound. Taken together, these latter results indicate that the R enantiomer is an antagonist of S isomer-promoted topoisomerase II-mediated DNA cleavage. Finally, the cytotoxic potential of quinolyl-substituted ofloxacin analogs correlated with the ability to stimulate enzyme-mediated DNA cleavage. Thus, stereochemistry appears to be a governing factor for the potential development of tricyclic quinolones as topoisomerase II-targeted drugs with antineoplastic activity.

The kinetics of oxidation of eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse ferrocytochromes c, modified at lysine 7, 13, 25, 27, 60, 72, 86, or 87, and of one trinitrophenyl horse ferrocytochrome c, modified at lysine 13, by the 3- and 3+ inorganic complexes hexacyanoferrate(III) (Fe(CN)6(3-)) and tris(1,10-phenanthroline)cobalt(III) (Co(phen)3(3+)) have been characterized. The influence of the modified residues on the bimolecular rate constants for these reactions define the protein molecular surface involved. The site of electron exchange for both oxidants appears to be the solvent accessible edge of the heme prosthetic group or a closely related structure on the "front" surface of the molecule. The reaction with Fe(CN)6(3-) is most strongly influenced by modification of lysine 72, a residue to the left of the exposed heme edge. (CDNP lysine 72 cytochrome c yields a 3.6-fold decrease in the bimolecular rate constant, as compared to that for the native protein.) However, it is the region around lysine 27, to the right of the heme edge, that is most influential in the reaction with Co(phen)3(3+). (CDNP-lysine 27 cytochrome c exhibits a 7.3-fold increase in the rate constant, as compared to that for the native protein.) The kinetics of reaction of the CDNP-lysine 13, 60, 72, and 87 modified cytochromes c with Fe(CN)5(4-aminopyridine)2- as oxidant and Fe(CN)5(4-aminopyridine)3- and Fe(CN)5-(imidazole)3- as reductants have also been determined and further illustrate the influence of electrostatics on the kinetics of such protein-small molecule electron exchanges.