DNA topological context affects access to eukaryotic DNA topoisomerase I

We have analyzed the reactivity of a 217 base pair segment of the intrinsically curved Crithidia fasciculata kinetoplast DNA towards eukaryotic DNA topoisomerase I. The substrates were open [linear fragment and nicked circle] and closed minidomains [closed relaxed circle and circles with linking differences of -1 and -2]. We interpreted the results with the aid of a model that was used to predict the structures of the topoisomers. The modelling shows that the delta Lk(-1) form is unusually compact because of the curvature in the DNA. To determine the role of sequence-directed curvature in both the experimental and modeling studies, controls were examined in which the curved Crithidia sequence was replaced by an uncurved sequence obtained from the plasmid pBR322. Reactivity of the Crithidia DNA [as analyzed both by the cleavage and topoisomerization reactions] markedly varied among the DNA forms: (i) the hierarchy of overall reactivity observed is: linear fragment > nicked circular, closed circular [delta Lk(0)], interwound [delta Lk(-2)] > bent interwound [delta Lk(-1)]; (ii) the intensity of several cleavage positions differs among DNA forms. The results show that eukaryotic DNA topoisomerase I is very sensitive to the conformation of the substrates and that its reactivity is modulated by the variation of the compactness of the DNA molecule. The C. fasciculata sequence contains a highly curved segment that determines the conformation of the closed circle in a complex way.     

A catalytic domain of eukaryotic DNA topoisomerase I

Eukaryotic type IB topoisomerases catalyze the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The 314-amino acid vaccinia topoisomerase is the smallest member of this family and is distinguished from its cellular counterparts by its specificity for cleavage at the target sequence 5'-CCCTT downward arrow. Here we show that Topo-(81-314), a truncated derivative that lacks the N-terminal domain, performs the same repertoire of reactions as the full-sized topoisomerase: relaxation of supercoiled DNA, site-specific DNA transesterification, and DNA strand transfer. Elimination of the N-terminal domain slows the rate of single-turnover DNA cleavage by 10(-3.6), but has little effect on the rate of single-turnover DNA religation. DNA relaxation and strand cleavage by Topo-(81-314) are inhibited by salt and magnesium; these effects are indicative of reduced affinity in noncovalent DNA binding. We report that identical properties are displayed by a full-length mutant protein, Topo(Y70A/Y72A), which lacks two tyrosine side chains within the N-terminal domain that contact the DNA target site in the major groove. We speculate that Topo-(81-314) is fully competent for transesterification chemistry, but is compromised with respect to a rate-limiting precleavage conformational step that is contingent on DNA contacts made by Tyr-70 and Tyr-72.     

Characterization of an altered DNA catalysis of a camptothecin-resistant eukaryotic topoisomerase I

We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.     

Polynucleotide ligase activity of eukaryotic topoisomerase I

1. Extrapyramidal adverse effects have been reported with the selective serotonin re-uptake inhibitor (SSRI) antidepressants, particularly fluoxetine and paroxetine. 2. Recently, the SSRI sertraline has also been associated with treatment-emergent extrapyramidal syndrome (EPS) side effects. A review of the literature identified thirteen published cases of sertraline-induced EPS, several of which were confounded by the presence of concomitant medications, and few reported quantitative data using rating scales. 3. We present another case of EPS associated with sertraline in which daily ratings were obtained using the Abnormal Involuntary Movement Scale. 4. This review and case report add to the small but growing literature suggesting that sertraline, like the other SSRI's may cause significant extrapyramidal side-effects. These movement disorders presumably occur through an interaction between serotonergic and dopaminergic pathways, providing an important clinical correlation of interactions between these neurotransmitter systems.     

The dihydropyridine dexniguldipine hydrochloride inhibits cleavage and religation reactions of eukaryotic DNA topoisomerase I

Dexniguldipine hydrochloride (B859-35, a dihydropyridine with antitumor and multidrug resistance-reverting activity) inhibits both the DNA cleavage and religation reactions of purified human DNA topoisomerase I at concentrations >1 microM, whereas at concentrations <1 microM it inhibits selectively the religation step and stabilizes the covalent topoisomerase I-DNA intermediate in a similar fashion as camptothecin. Inhibition of religation by camptothecin can be overcome by increasing the concentration of the DNA substrate in the religation reaction, indicating a competitive type of inhibition. In contrast, dexniguldipine hydrochloride decreases rate constants of topoisomerase I-mediated DNA religation independently of the concentration of the DNA substrate, suggesting a noncompetitive mechanism of inhibition, which is different from that of camptothecin.     

Mitochondrial DNA topoisomerase I of Saccharomyces cerevisiae

The radiation protective effect of thiol compounds is unequivocal and their use is only limited by their toxic effects. We used the principle of alpha alkylation, which renders amino acids unmetabolizable, to reduce the toxicity of homocysteine. This product, alpha-methyl-homocysteine thio-lactone, was tested for toxicity and radiation protective effect along with known protectors L-cysteine, cysteamine and WR 1065 in cell culture using V79-4 Chinese hamster lung cells. The three-day growth curve assays, useful to measure overall effects on cell growth, revealed lowest toxicity for alpha-methyl-homocysteine thiolactone (GL-2). Clonogenic survival tests, used to evaluate the retention of reproductive integrity, were carried out and revealed that GL-2 had no adverse effects in this test system. Radiation protection tests showed that GL-2 exhibited protective activity against radiation induced lethality above that seen with cysteine and cysteamine, but below WR 1065. However, GL-2 showed little or no negative effects toward the cell itself, in direct contrast to WR 1065. Our findings show a potentially important tool and principle to reduce toxicity of radiation protectors with analogous structures.     

Vaccinia virus DNA topoisomerase: a model eukaryotic type IB enzyme

Vaccinia topoisomerase has proven to be an instructive model system for mechanistic studies of the type IB family of DNA topoisomerases. The catalytically relevant functional groups at the active site and the circumferential topoisomerase-DNA interface were correctly surmised by mutational and footprint analysis of vaccinia topoisomerase in advance of structure determinations by X-ray crystallography. It is now evident from multiple crystal structures that the catalytic domains of type IB topoisomerases and site specific recombinases derive from a common ancestral strand transferase capable of forming a DNA-(3'-phosphotyrosyl)-enzyme intermediate. A constellation of conserved amino acids catalyzes attack of the tyrosine nucleophile on the scissile phosphate. Domain dynamics and DNA-induced conformational changes within the catalytic domain are likely to play a role in triggering strand scission and coordinating the strand exchange or strand passage steps.     

Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases

OBJECTIVE: The goals were to summarize the results of liver transplantation for chronic hepatitis B disease (HBV) at the University of Virginia, correlate pretransplant viral markers with posttransplant hepatitis B immunoglobulin (HBIg) requirements, and identify the relation between viral protein in the liver and clinical reinfection. SUMMARY BACKGROUND DATA: Liver transplantation is an accepted treatment for end-stage liver disease from chronic HBV infection, although lifelong antiviral treatment (with HBIg or antiviral agents) is still necessary. Patients with evidence of active viral replication (detectable serum HBV-DNA or e antigen) at the time of transplant have a higher rate of allograft infection. Whether clinically stable patients receiving HBIg immunoprophylaxis have detectable viral products in their grafts remains unknown. METHODS: Forty-four transplants performed for HBV disease at the University of Virginia since March 1990 were reviewed. Most patients underwent aggressive passive immunoprophylaxis with HBIg to maintain serum HBV surface antibody (HBsAb) levels > or =500 IU/l for the first 6 months after the transplant, and > or =150 IU/l thereafter. Patients had viral markers quantified, underwent pharmacokinetic analysis of HBsAb levels to adjust dosing, and were biopsied routinely every 3 to 6 months and when indicated. RESULTS: Forty-four transplants were performed in 39 patients. Actual 1-year and 3-year graft survival was 95% and 81%, respectively, and 1-year and 3-year patient survival was 98% and 96%, respectively. After the adoption of indefinite HBIg prophylaxis, nine grafts became infected (all in recipients positive for HBV e antigen). Three occurred within 8 weeks of transplantation and were associated with a short HBsAb half-life and a wild-type virus. Six occurred >8 months after the transplant, and most of these were associated with viral mutation. Quantification of pretransplant markers was an overall poor predictor of HBIg requirements after the transplant. Immunohistochemistry demonstrated transient low-level expression of core protein in the liver in 23% of patients without serum or clinical evidence of recurrent hepatitis. CONCLUSIONS: An excellent outcome is possible after liver transplantation for chronic HBV disease using HBIg dosed by pharmacokinetic parameters. Currently, quantification of pretransplant serum markers of the HBV antigen load does not predict the intensity of posttransplant treatment required for good clinical outcomes. Because HBV is not eradicated from the patient, some form of indefinite antiviral therapy continues to be warranted.     

Physiological regulation of eukaryotic topoisomerase II

Topoisomerase II is an essential enzyme in all organisms with several independent roles in DNA metabolism. In this article we review our knowledge on the regulation of the expression and catalytic activity of topoisomerase II in both lower and higher eukaryotes. Current data indicate that the regulation of topoisomerase II gene expression is complex, with positive and negative controls in evidence at the level of both promoter activity and mRNA stability. Similarly, the activity of the mature enzyme can be regulated by the action of several different protein kinases. Of particular interest is the cell cycle-dependent phosphorylation of topoisomerase II, including multiple, mitosis-specific modifications, which are proposed to regulate the essential chromosome decatenation activity of the enzyme.     

Expression of DNA topoisomerase I, DNA topoisomerase II-alpha, and p53 in metastatic malignant melanoma

New anticancer drugs that target DNA topoisomerase I (topo I) are showing activity against a wide variety of solid human neoplasms. These drugs work by a novel mechanism of action and cause topo I-mediated DNA breaks. These DNA breaks become lethal in cycling cells when they interact with the replication fork. Because of the challenges in treating metastatic malignant melanoma, we performed an immunohistochemical study of this group of neoplasms to search for the presence of molecular markers that might indicate tumor response to topo I active drugs. Using a new immunohistochemical stain for topo I, we found elevation of this protein in 10 of 24 cases (41.6%) of metastatic malignant melanoma. The metastatic tumors that showed increased expression of topo I (2+ or 3+) had statistically significant higher proliferation indices, measured by immunohistochemical staining for DNA topo II-alpha, than did metastatic lesions with no detectable topo I expression. The average topo II-alpha index of metastatic melanomas with 2+ topo I expression was 45.1 (SD = 17.9) and with 3+ topo I expression was 52.3 (SD = 32.5). These values were found to be statistically different (P = .05) than the average topo II-alpha index of 18.9 (SD = 17.7) found for metastatic melanomas without detectable topo I immunostaining. Immunohistochemical staining for p53 suggested abnormal p53 function in 6 of the 10 melanomas (60%), which showed elevations of topo I (2 to 3+ topo I immunostaining) but normal p53 function in all 14 metastatic lesions that showed normal topo I expression.     

The ability of thiram to inhibit eukaryotic topoisomerase II and to damage DNA

At the end of a hybridoma batch culture, the cells are usually discarded after separation from the culture broth. If, however, they are aseptically recycled into the reactor, the production process can be resumed simply by the addition of fresh medium. This cycle can then be repeated several times consecutively. In a test case, with a mouse hybridoma, we found antibody yields for each cycle in the same range as for a standard batch. In a 15 1 stirred tank reactor we could, within 6 days, produce 2.8 g of monoclonal antibody (MAb). This type of reactor operation allowed a doubling in the reactor volumetric productivity (mg/l/day).     

Rhodobacter capsulatus DNA topoisomerase I purification and characterization

A 30-kDa DNA topoisomerase has been purified to near homogeneity from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus. The enzyme is recognized by an antibody against a 16-mer peptide sequence from human DNA topoisomerase I. The purified enzyme is a type I topoisomerase. Consistent with the properties of other prokaryotic type I DNA topoisomerases, the isolated enzyme is unable to relax positively supercoiled DNA and absolutely requires divalent cations for its relaxation activity. However, regardless of the Mg+2 concentrations, ATP concentrations above 5 mM completely inhibit the relaxing activity. The enzyme is sensitive to high salt concentrations and the optimal activity occurs at salt concentrations between 3 and 30 mM for monovalent cations. Single-stranded M13 DNA is a strong inhibitor of this relaxing activity. The enzyme is inhibited by ethidium bromide, confirming that this DNA topoisomerase is incapable of relaxing positive supercoils. Topoisomerase I-specific inhibitors like Hoechst 32258 and actinomycin D inhibit the enzymatic activity while the enzyme is resistant to type II topoisomerase inhibitors such as norfloxacin, nalidixic acid, and novobiocin. From these enzymatic characteristics, we conclude that the R. capsulatus DNA topoisomerase is a prokaryotic type I DNA topoisomerase.     

DNA minor groove binding-directed poisoning of human DNA topoisomerase I by terbenzimidazoles

We have employed a broad range of spectroscopic, calorimetric, DNA cleavage, and DNA winding/unwinding measurements to characterize the DNA binding and topoisomerase I (TOP1) poisoning properties of three terbenzimidazole analogues, 5-phenylterbenzimidazole (5PTB), terbenzimidazole (TB), and 5-(naphthyl[2,3-d]imidazo-2-yl)bibenzimidazole (5NIBB), which differ with respect to the substitutions at their C5 and/or C6 positions. Our results reveal the following significant features. (i) The overall extent to which the three terbenzimidazole analogues poison human TOP1 follows the hierarchy 5PTB > TB > 5NIBB. (ii) The impact of the three terbenzimidazole analogues on the superhelical state of plasmid DNA depends on the [total ligand] to [base pair] ratio (rbp), having no effect on DNA superhelicity at rbp ratios < or = 0.1, while weakly unwinding DNA at rbp ratios > 0.1. This weak DNA unwinding activity exhibited by the three terbenzimidazoles does not appear to be correlated with the abilities of these compounds to poison TOP1. (iii) Upon complexation with both poly(dA).poly(dT) and salmon testes DNA, the three terbenzimidazole analogues exhibit flow linear dichroism properties characteristic of a minor groove-directed mode of binding to these host DNA duplexes. (iv) The apparent minor groove binding affinities of the three terbenzimidazole analogues for the d(GA4T4C)2 duplex follow a qualitatively similar hierarchy to that noted above for ligand-induced poisoning of human TOP1-namely, 5PTB > TB > 5NIBB. In the aggregate, our results suggest that DNA minor groove binding, but not DNA unwinding, is important in the poisoning of TOP1 by terbenzimidazoles.     

An inhibitor of DNA topoisomerase I from Xenopus laevis ovaries

A novel, heat-resistant and Pronase-sensitive, inhibitor of eukaryotic DNA topoisomerase I has been purified from Xenopus laevis ovaries. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the most purified fraction revealed three bands with apparent molecular masses of 25, 28.5, and 33.5 kDa. The 25- and 33.5-kDa peptides recovered from an SDS-PAGE gel inhibited X. laevis DNA topoisomerase I. The purified inhibitor was specific to DNA topoisomerase I and did not inhibit other DNA enzymes tested. The inhibitor blocked the catalytic activity of DNA topoisomerase I by interacting with the enzyme, rather than by competing for binding sites on substrate DNA. Binding of DNA topoisomerase I to substrate DNA was blocked by the inhibitor, as was the cleavage reaction catalyzed by DNA topoisomerase I. Inhibition of DNA topoisomerase I was relieved by divalent cations Ca2+, Mg2+, or Mn2+.     

Inhibitors of DNA topoisomerase I and II isolated from the Coptis rhizomes

A space-occupying lesion 3.5 by 2.0 cm in size caused by Capillaria infection was revealed ultrasonographically in segment 6 (S6) of the liver of a 32-year-old woman from Okinawa, Japan, who was hospitalized with a complaint of pain in the right upper quadrant. Laboratory examination showed leukocytosis of 10,400/mm3 with 22% eosinophils and slight impairment of liver function. The tumor was removed surgically and found to be a necrotic granuloma with eosinophilic infiltration formed around a degenerated nematode. The causative agent was presumed to be Capillaria hepatica based on the morphology of the bacillary bands and stichosome observed in the sectioned worm and in the fragments of worm recovered by dissecting the tumor tissue that was embedded in paraffin.     

Mutations of vaccinia virus DNA topoisomerase I that stabilize the cleavage complex

Two mutations in vaccinia virus topoisomerase I, K167D and G226N, have been characterized. SOS induction was observed in Escherichia coli expressing vaccinia topoisomerase I with either one of these mutations. The mutant enzymes were purified to homogeneity and compared with the wild type enzyme for relaxation activity and the partial activities of substrate binding, site-specific DNA cleavage and DNA religation to determine the mechanism of SOS induction. The K167D mutant enzyme had reduced binding affinity for the DNA substrate with a Kapp that was 10-fold higher than wild type. Nevertheless, in reactions with high enzyme concentration, its substrate cleavage activity was 90% that of wild type. The G226N mutant enzyme had virtually wild type binding and cleavage activities. However, intermolecular religation by these two mutants were observed to be significantly reduced. The cleavage complexes formed with the K167D and G226N mutants were more stable to high salt than the wild type cleavable complex. We propose that these mutants in vivo induce the SOS response in E. coli due to the shift of topoisomerase cleavage-religation equilibrium towards cleavage and increased stability of the cleavage complex. The mutation thus has a similar effect as the topoisomerase-targeting inhibitors that turn topoisomerases into DNA damaging agents.     

Caspase-mediated cleavage of DNA topoisomerase I at unconventional sites during apoptosis

Previous studies have demonstrated that topoisomerase I is cleaved late during apoptosis, but have not identified the proteases responsible or examined the functional consequences of this cleavage. Here, we have shown that treatment of purified topoisomerase I with caspase-3 resulted in cleavage at DDVD146 downward arrowY and EEED170 downward arrowG, whereas treatment with caspase-6 resulted in cleavage at PEDD123 downward arrowG and EEED170 downward arrowG. After treatment of Jurkat T lymphocytic leukemia cells with anti-Fas antibody or A549 lung cancer cells with topotecan, etoposide, or paclitaxel, the topoisomerase I fragment comigrated with the product that resulted from caspase-3 cleavage at DDVD146 downward arrowY. In contrast, two discrete topoisomerase I fragments that appeared to result from cleavage at DDVD146 downward arrowY and EEED170 downward arrowG were observed after treatment of MDA-MB-468 breast cancer cells with paclitaxel. Topoisomerase I cleavage did not occur in apoptotic MCF-7 cells, which lack caspase-3. Cell fractionation and band depletion studies with the topoisomerase I poison topotecan revealed that the topoisomerase I fragment remains in proximity to the chromatin and retains the ability to bind to and cleave DNA. These observations indicate that topoisomerase I is a substrate of caspase-3 and possibly caspase-6, but is cleaved at sequences that differ from those ordinarily preferred by these enzymes, thereby providing a potential explanation why topoisomerase I cleavage lags behind that of classical caspase substrates such as poly(ADP-ribose) polymerase and lamin B1.     

Effects of DNA methylation on topoisomerase I and II cleavage activities

DNA methylation is deregulated during oncogenesis. Since several major anti-cancer drugs act on topoisomerases, we investigated the effects of cytosine methylation on topoisomerase cleavage activities. Both topoisomerase I and II cleavage patterns were modified by CpG methylation in c-myc gene DNA fragments. Topoisomerase II changes, mainly cleavage reduction, occurred for methylation sites within 7 base pairs from the topoisomerase II breaks and were different for VM-26 and azatoxin. For topoisomerase I, cleavage enhancement as well as suppression were observed. Using synthetic methylated oligonucleotides, we show that hemimethylation is sufficient to alter topoisomerase I activity. Cytosine methylation on the scissile strand within the topoisomerase I consensus sequence had strong effects. Cleavage was stimulated by methylation at position -4 and was strongly inhibited by methylation at position -3 (with position -1 being the enzyme-linked nucleotide). This inhibitory effect was attributed to the presence of a methyl group in the major groove, since the transition uracil to thymine also inhibited cleavage. Altogether these results suggest an interaction of topoisomerase I with the DNA major grove at positions -3 and -4. In addition, DNA methylation may have profound effects on the activity of topoisomerases and may alter the distribution of cleavage sites produced by anticancer drugs in chromatin.