Search

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.

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.

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.