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1.
DNA topoisomerase II is an essential nuclear enzyme for proliferation of eukaryotic cells and plays important roles in many aspects of DNA processes. In this report, we have demonstrated that the catalytic activity of topoisomerase IIalpha, as measured by decatenation of kinetoplast DNA and by relaxation of negatively supercoiled DNA, was stimulated approximately 2-3-fold by the tumor suppressor p53 protein. In order to determine the mechanism by which p53 activates the enzyme, the effects of p53 on the topoisomerase IIalpha-mediated DNA cleavage/religation equilibrium were assessed using the prototypical topoisomerase II poison, etoposide. p53 had no effect on the ability of the enzyme to make double-stranded DNA break and religate linear DNA, indicating that the stimulation of the enzyme catalytic activity by p53 was not due to alteration in the formation of covalent cleavable complexes formed between topoisomerase IIalpha and DNA. The effects of p53 on the catalytic inhibition of topoisomerase IIalpha were examined using a specific catalytic inhibitor, ICRF-193, which blocks the ATP hydrolysis step of the enzyme catalytic cycle. Clearly manifested in decatenation and relaxation assays, p53 reduced the catalytic inhibition of topoisomerase IIalpha by ICRF-193. ATP hydrolysis assays revealed that the ATPase activity of topoisomerase IIalpha was specifically enhanced by p53. Immunoprecipitation experiments revealed that p53 physically interacts with topoisomerase IIalpha to form molecular complexes without a double-stranded DNA intermediary in vitro. To investigate whether p53 stimulates the catalytic activity of topoisomerase II in vivo, we expressed wild-type and mutant p53 in Saos-2 osteosarcoma cells lacking functional p53. Wild-type, but not mutant, p53 stimulated topoisomerase II activity in nuclear extract from these transfected cells. Our data propose a new role for p53 to modulate the catalytic activity of topoisomerase IIalpha. Taken together, we suggest that the p53-mediated response of the cell cycle to DNA damage may involve activation of topoisomerase IIalpha.  相似文献   

2.
Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II was studied in the absence and presence of DNA, and in the absence and presence of inhibitor ICRF-193. The results indicate that purified Top2-(1-409), a fragment containing the NH(2)-terminal 409 amino acids of the yeast enzyme, is predominantly monomeric, with a low level of ATPase owing to weak association of two monomers to form a catalytically active dimer. The ATPase activity of Top2-(1-409) is independent of DNA in a buffer containing 100 mM NaCl, in which intact yeast DNA topoisomerase II exhibits robust DNA-dependent ATPase and DNA transport activities. Purified Top2-(1-660), a fragment containing the NH(2)-terminal 660 amino acid of the yeast enzyme, appears to be dimeric in the absence or presence of DNA, and the ATPase activity of the protein is significantly stimulated by DNA. These results are consistent with a model in which binding of an intact DNA topoisomerase II to DNA places the various subfragments of the enzyme in a way that makes the intramolecular dimerization of the ATPase domains more favorable. We believe that this alignment of subfragments is mainly achieved through the binding of the enzyme to the DNA segment within which the enzyme makes transient breaks. The ATPase activity of Top2-(1-409) is inhibited by ICRF-193, suggesting that the bisdioxopiperazine class of DNA topoisomerase II inhibitors directly interacts with the paired ATPase domains of the enzyme.  相似文献   

3.
Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerase-targeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a cytotoxic DNA-damaging agent. Here we report that ICRF-193 is a very significant topoisomerase II poison. Detection of topoisomerase II poisoning by ICRF-193 required the use of a chaotropic protein denaturant in the topoisomerase poisoning assays. ICRF-193 caused dose-dependent cross-linking of human topoisomerase IIbeta to DNA and stimulated topoisomerase IIbeta-mediated DNA cleavage at specific sites on (32)P-end-labeled DNA. Human topoisomerase IIalpha-mediated DNA cleavage was stimulated to a lesser extent by ICRF-193. In vivo experiments with MCF-7 cells also showed the requirement of a chaotropic protein denaturant in the assays and selectivity for the beta-isozyme of human topoisomerase II. Studies with two topoisomerase IIbeta-negative cell model systems confirmed significant topoisomerase II poisoning by ICRF-193 in the wild type cells and were consistent with beta-isozyme selectivity. Common use of only the detergent, SDS, in assays may have led to failure to detect topoisomerase II poisoning by ICRF-193 in earlier studies.  相似文献   

4.
5.
The bis-dioxopiperazine ICRF-193 has long time been considered as a pure topoisomerase II catalytic inhibitor able to exert its inhibitory effect on the enzyme without stabilization of the so-called cleavable complex formed by DNA covalently bound to topoisomerase II. In recent years, however, this concept has been challenged, as a number of reports have shown that ICRF-193 really "poisons" the enzyme, most likely through a different mechanism from that shown by the classical topoisomerase II poisons used in cancer chemotherapy. In the present investigation, we have carried out a study of the capacity of ICRF-193 to induce DNA strand breaks, as classical poisons do, in cultured V79 and irs-2 Chinese hamster lung fibroblasts using the comet assay and pulsed-field gel electrophoresis (PFGE). Our results clearly show that ICRF-193 readily induces breakage in DNA through a mechanism as yet poorly understood.  相似文献   

6.
It has been proposed that xanthone derivatives with anticancer potential act as topoisomerase II inhibitors because they interfere with the ability of the enzyme to bind its ATP cofactor. In order to further characterize xanthone mechanism and generate compounds with potential as anticancer drugs, we synthesized a series of derivatives in which position 3 was substituted with different polyamine chains. As determined by DNA relaxation and decatenation assays, the resulting compounds are potent topoisomerase IIα inhibitors. Although xanthone derivatives inhibit topoisomerase IIα-catalyzed ATP hydrolysis, mechanistic studies indicate that they do not act at the ATPase site. Rather, they appear to function by blocking the ability of DNA to stimulate ATP hydrolysis. On the basis of activity, competition, and modeling studies, we propose that xanthones interact with the DNA cleavage/ligation active site of topoisomerase IIα and inhibit the catalytic activity of the enzyme by interfering with the DNA strand passage step.  相似文献   

7.
DNA topoisomerase II catalyzes the transport of one DNA duplex through a transient break in a second duplex using a complex ATP hydrolysis mechanism. Two key rates in the ATPase mechanism, ATP resynthesis and phosphate release, were investigated using 18O exchange and stopped-flow phosphate release experiments, respectively. The 18O exchange results showed that the rate of ATP resynthesis on the topoisomerase II active site was slow compared with the rate of phosphate release. When topoisomerase II was bound to DNA, phosphate was released slowly, with a lag. Since each of the preceding steps is known to occur rapidly, phosphate release is apparently a rate-determining step. The length of the lag phase was unaffected by etoposide, indicating that inhibiting DNA religation inhibits the ATPase reaction cycle at some step following phosphate release. By combining the 18O exchange and phosphate release results, the rate constant for ATP resynthesis can be calculated as approximately 0.5 s(-1). These data support the mechanism of sequential hydrolysis of two ATP by DNA topoisomerase II.  相似文献   

8.
The catalytic activity of topoisomerase II is stimulated approximately 2-3-fold following phosphorylation by casein kinase II (Ackerman, P., Glover, C. V. C., and Osheroff, N. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3164-3168). In order to delineate the mechanism by which the activity of the enzyme is enhanced, the effects of casein kinase II-mediated phosphorylation on the individual steps of the catalytic cycle of Drosophila topoisomerase II were characterized. Phosphorylation did not affect reaction steps that preceded hydrolysis of the enzyme's high energy ATP cofactor. This included enzyme-DNA binding, pre-strand passage DNA cleavage/religation, the double-stranded DNA passage event, and post-strand passage DNA cleavage/religation. In contrast, the rate of topoisomerase II-mediated ATP hydrolysis was stimulated 2.7-fold following phosphorylation by casein kinase II. Since ATP hydrolysis is a prerequisite for enzyme turnover, it is concluded that phosphorylation modulates the overall catalytic activity of topoisomerase II by stimulating the enzyme's ATPase activity.  相似文献   

9.
Topoisomerase II is an essential enzyme that is targeted by a number of clinically valuable anticancer drugs. One class referred to as topoisomerase II poisons works by increasing the cellular level of topoisomerase II-mediated DNA breaks, resulting in apoptosis. Another class of topoisomerase II-directed drugs, the bis-dioxopiperazines, stabilizes the conformation of the enzyme where it attains an inactive salt-stable closed clamp structure. Bis-dioxopiperazines, similar to topoisomerase II poisons, induce cell killing, but the underlying mechanism is presently unclear. In this study, we use three different biochemically well characterized human topoisomerase IIalpha mutant enzymes to dissect the catalytic requirements needed for the enzyme to cause dominant sensitivity in yeast to the bis-dioxopirazine ICRF-193 and the topoisomerase II poison m-AMSA. We find that the clamp-closing activity, the DNA cleavage activity, and even both activities together are insufficient for topoisomerase II to cause dominant sensitivity to ICRF-193 in yeast. Rather, the strand passage event per se is an absolute requirement, most probably because this involves a simultaneous interaction of the enzyme with two DNA segments. Furthermore, we show that the ability of human topoisomerase IIalpha to cause dominant sensitivity to m-AMSA in yeast does not depend on clamp closure or strand passage but is directly related to the capability of the enzyme to respond to m-AMSA with increased DNA cleavage complex formation.  相似文献   

10.
We have prepared full-length Drosophila and human topoisomerase II and truncation constructs containing the amino-terminal ATPase domain, and we have analyzed their biochemical properties. The ATPase activity of the truncation proteins, similar to that of the full-length proteins, is greatly stimulated by the presence of DNA. This activity of the truncation proteins is also sensitive to the inhibition by the drug bisdioxopiperazine, ICRF-193, albeit at a much lower level than the full-length protein. Therefore, bisdioxopiperazine can directly interact with the NH(2)-terminal ATPase domain, but the drug-enzyme interaction may involve other domains as well. The ATPase activity of the ATPase domain protein showed a quadratic dependence on enzyme concentration, suggesting that dimerization of the NH(2)-terminal domain is a rate-limiting step. Using both protein cross-linking and sedimentation equilibrium analysis, we showed that the ATPase domain exists as a monomer in the absence of cofactors but can readily dimerize in the presence of a nonhydrolyzable analog of ATP, 5'-adenylyl-beta,gamma-imidodiphosphate. More interestingly, both ATP and ADP can also promote protein dimerization. This result thus suggests that the protein clamp, mediated through the dimerization of ATPase domain, remains closed after ATP hydrolysis and opens upon the dissociation of ADP.  相似文献   

11.
DNA topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double-stranded DNA segment and transporting it through another duplex. Surface plasmon resonance (SPR) was used to characterize interactions of human topoisomerase II alpha with different topological forms of DNA. Using a linear fragment of pUC18 DNA, the equilibrium binding constant of topoisomerase II alpha was determined to be 0.16 nM. The affinity was not affected by the absence of ATP or the presence of the bisdioxopiperazine catalytic inhibitor ICRF-187. Besides, similar affinities were found for several bisdioxopiperazine-resistant mutant enzymes. These results suggest that the mechanism of topoisomerase II alpha inhibition by ICRF-187 and its resistance does not directly involve the interaction of DNA with the enzyme. SPR was also adapted to measure levels of the closed clamp form of topoisomerase II present on DNA. As expected, a stable closed clamp form of the enzyme was detectable on circular DNA but not on linear DNA. Detection of the closed clamp required the presence of ATP and a bisdioxopiperazine, or a non-hydrolyzable analogue of ATP. In the presence of ATP and ICRF-187, several bisdioxopiperazine-resistant mutant enzymes failed to form detectable levels of stable closed clamp. Interestingly, a mutant of human topoisomerase II alpha with an altered active site tyrosine showed lower levels of closed clamp formation. In conclusion, SPR is able to (1) determine the kinetics of topoisomerase II with its DNA substrate and (2) quantify the enzyme's closed clamp formation under varying circumstances.  相似文献   

12.
With the ultimate purpose of testing the existence of possible differences in the effectiveness of the topoisomerase II catalytic inhibitor ICRF-193 (a bisdioxopiperazine) and the enzyme suppressor bufalin (a bufadienolide from toad venom) we have carried out a series of experiments aimed at inducing cytotoxicity as well as DNA and chromosome damage in transformed CHO cells. In order to assess any possible influence of DNA repair capacity of the treated cells on the final outcome, we have made use of the repair-defective CHO mutant EM9, which shows a defect in DNA single- and double-strand breaks repair for comparison with its repair-proficient parental line AA8.Our results seem to indicate that, while both ICRF-193 and bufalin suppress cell growth and result in a clear inhibition of topoisomerase II catalytic activity, only ICRF-193 has been shown as able to induce both chromosome and DNA damage, with a more pronounced effect in the CHO mutant EM9 than in the repair-proficient line AA8.  相似文献   

13.
Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, on the basis of the reactivity changes of several cysteines located at the N-gate and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5'-adenylyl β,γ-imidodiphosphate or the anticancer drug ICRF-193.  相似文献   

14.
Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving gamma-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation.  相似文献   

15.
DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclin-dependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.

Key Words:

Plant cyclin B2, Topoisomerase II, ICRF-193, G2 checkpoint, Microtubules  相似文献   

16.
Eukaryotic DNA topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. It changes the topology of DNA by coupling binding and hydrolysis of two ATP molecules to the transport of one DNA duplex through a temporary break introduced in another. During this process the structurally and functionally complex enzyme passes through a cascade of conformational changes, which requires intra- and intersubunit communication. To study the importance of ATP binding and hydrolysis in relation to DNA strand transfer, we have purified and characterized a human topoisomerase II alpha heterodimer with only one ATP binding site. The heterodimer was able to relax supercoiled DNA, although less efficiently than the wild type enzyme. It furthermore possessed a functional N-terminal clamp and was sensitive to ICRF-187. This demonstrates that human topoisomerase II alpha can pass through all the conformations required for DNA strand passage and enzyme resetting with binding and hydrolysis of only one ATP. However, the heterodimer lacked the normal stimulatory effect of DNA on ATP binding and hydrolysis as well as the stimulatory effect of ATP on DNA cleavage. The results can be explained in a model, where efficient catalysis requires an extensive communication between the second ATP and the DNA segment to be cleaved.  相似文献   

17.
Topoisomerase II-catalyzed DNA transport requires coordination between two distinct reactions: ATP hydrolysis and DNA cleavage/religation. To further understand how these reactions are coupled, inhibition by the clinically used anticancer drug etoposide was studied. The IC(50) for perturbing the DNA cleavage/religation equilibrium is nucleotide-dependent; its value is 6 microM in the presence of ATP, 25 microM in the presence of a nonhydrolyzable ATP analog, and 45 microM in the presence of ADP or no nucleotide. This inhibition was further characterized using steady-state and pre-steady-state ATPase and decatenation assays. Etoposide is a hyperbolic noncompetitive inhibitor of the ATPase activity with a K(i)(app) of 5.6 microM no inhibition of ATP hydrolysis is seen in the absence of DNA cleavage. In order to determine which steps of the ATPase mechanism etoposide inhibits, pre-steady-state analysis was performed. These results showed that etoposide does not reduce the rate of binding two ATP, hydrolyzing the first ATP, or releasing the second ADP. Inhibition is therefore associated with the first product release step or hydrolysis of the second ATP, suggesting that DNA religation normally occurs at one of these two steps. Multiple turnover decatenation is inhibited when etoposide is present; however, single turnover decatenation occurs normally. The implications of these results are discussed in terms of their contribution to our current model for the topoisomerase II mechanism.  相似文献   

18.
An ATR-dependent G(2) checkpoint responds to inhibition of topoisomerase II and delays entry into mitosis by sustaining nuclear exclusion of cyclin B1-Cdk1 complexes. Here we report that induction of this checkpoint with ICRF-193, a topoisomerase II catalytic inhibitor that does not cause DNA damage, was associated with an ATR-dependent inhibition of polo-like kinase 1 (Plk1) kinase activity and a decrease in cyclin B1 phosphorylation. Expression of constitutively active Plk1 but not wild type Plk1 reversed ICRF-193-induced mitotic delay in HeLa cells, suggesting that Plk1 kinase activity is important for the checkpoint response to ICRF-193. G(2)/M synchronized normal human fibroblasts, when treated with ICRF-193, showed a decrease in cyclin B1 phosphorylation and Plk1 kinase activity despite high cyclin B1-Cdk1 kinase activity. G(2) fibroblasts that were treated with caffeine to override the checkpoint response to ICRF-193 displayed a high incidence of chromosomal aberrations. Taken together, these results suggest that ATR-dependent inhibition of Plk1 kinase activity may be one mechanism to regulate cyclin B1 phosphorylation and sustain nuclear exclusion during the G(2) checkpoint response to topoisomerase II inhibition. Moreover, the results demonstrate an important role for the topoisomerase II-dependent G(2) checkpoint in the preservation of human genomic stability.  相似文献   

19.
Topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double stranded DNA segment and transporting it through another DNA molecule. Despite the extensive use of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, much remains unknown concerning the interactions between these agents and topoisomerase II. To identify the interaction of the bisdioxopiperazine dexrazoxane (ICRF-187) with topoisomerase II, we developed a rapid gel-filtration assay and characterized the binding of ((3)H)-dexrazoxane to human topoisomerase II alpha. Dexrazoxane binds to human topoisomerase II alpha in the presence of DNA and ATP with an apparent K(d) of 23 microM and a stoichiometry of 1 drug molecule per enzyme dimer. Various N-terminal single amino acid substitutions in human topoisomerase II alpha that were previously shown to confer specific bisdioxopiperazine resistance either totally abolished drug binding or resulted in less efficient binding. The effect of the various mutations on drug binding correlated well with their effect on drug resistance in vivo and in vitro. Interestingly, an altered active site tyrosine mutant of human topoisomerase II alpha, which is incapable of carrying out DNA strand passage, was unable to bind dexrazoxane, which agrees with the drug's proposed mechanism of action late in the topoisomerase II catalytic cycle. The direct correlation between the level of drug binding and dexrazoxane resistance is consistent with a decreased drug binding mechanism of action for these dexrazoxane resistance conferring mutations.  相似文献   

20.
DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclindependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.  相似文献   

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