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.     

DNA tridimensional context affects the reactivity of eukaryotic DNA topoisomerase I

Cleavage sites of eukaryotic DNA topoisomerase I on curved linear DNAs are clustered, map on the same side of the curve (the external one) and their distribution has the same period as the helical repeat, as observed on curved DNA tracts of Crithidia fasciculata, of Saccharomyces cerevisiae ARS1, of pT7CAT and on synthetic DNAs. The effects of the tridimensional context on both the cleavage and the topoisomerization reactions of DNA topoisomerase I were determined using serial DNA constructs made with inserts in which synthetic curves lie in a plane and in which the orientation of the planes of curvature is shifted by 72 degrees, 144 degrees, 216 degrees, 288 degrees and 360 degrees. The insertion of a curve markedly changes the reaction properties of the surrounding sequences.     

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.     

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.     

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).     

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.     

2.3 A crystal structure of the catalytic domain of DNA polymerase beta

The crystal structure of the catalytic domain of rat DNA polymerase beta (pol beta) has been determined at 2.3 A resolution and refined to an R factor of 0.22. The mixed alpha/beta protein has three subdomains arranged in an overall U shape reminiscent of other polymerase structures. The folding topology of pol beta, however, is unique. Two divalent metals bind near three aspartic acid residues implicated in the catalytic activity. In the presence of Mn2+ and dTTP, interpretable electron density is seen for two metals and the triphosphate, but not the deoxythymidine moiety. The principal interaction of the triphosphate moiety is with the bound divalent metals.     

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.     

Mutational analysis of Chlorella virus DNA ligase: catalytic roles of domain I and motif VI

A conserved catalytic core of the ATP-dependent DNA ligases is composed of an N-terminal domain (domain 1, containing nucleotidyl transferase motifs I, III, IIIa and IV) and a C-terminal domain (domain 2, containing motif VI) with an intervening cleft. Motif V links the two structural domains. Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the reaction of ligase with ATP to form ligase-adenylate, but is dispensable for the two subsequent steps in the ligation pathway; DNA-adenylate formation and strand closure. We find that formation of a phosphodiester at a pre-adenylated nick is subject to a rate limiting step that does not apply during the sealing of nicked DNA by ligase-adenylate. This step, presumably conformational, is accelerated or circumvented by deleting five amino acids of motif VI. The motif I lysine nucleophile (Lys27) is not required for strand closure by wild-type ligase, but this residue enhances the closure rate by a factor of 16 when motif VI is truncated. We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase--adenylate formation, but competent to catalyze strand closure at a pre-adenylated nick. These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction.     

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+.     

Merbarone inhibits the catalytic activity of human topoisomerase IIalpha by blocking DNA cleavage

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.     

Identification of mutations at DNA topoisomerase I responsible for camptothecin resistance

A camptothecin-resistant cell line that exhibits more than 600-fold resistance to camptothecin, designated CPT(R)-2000, was established from mutagen-treated A2780 ovarian cancer cells. CPT(R)-2000 cells also exhibit 3-fold resistance to a DNA minor groove-binding ligand Ho33342, a different class of mammalian DNA topoisomerase I inhibitors. However, CPT(R)-2000 cells exhibit no cross-resistance toward drugs such as Adriamycin, amsacrine, vinblastine, and 4'-dimethyl-epipodophyllotoxin. The mRNA, protein levels, and enzyme-specific activity of DNA topoisomerase I are relatively the same in parental and CPT(R)-2000 cells. However, unlike the DNA topoisomerase I activity of parental cells, which can be inhibited by camptothecin, that of CPT(R)-2000 cells cannot. In addition, parental cells after camptothecin treatment results in a decrease in the level of DNA topoisomerase I, whereas CPT(R)-2000 cells are insensitive to camptothecin treatment. These results suggested that the mechanism of camptothecin resistance is most likely due to a DNA topoisomerase I structural mutation. This notion is supported by DNA sequencing results confirming that DNA topoisomerase I of CPT(R)-2000 is mutated at amino acid residues Gly717 to Val and Thr729 to Ile. We also used the yeast system to examine the mutation(s) responsible for camptothecin resistance. Our results show that each single amino acid change results in partial resistance, and the double mutation gives a synergetic effect on camptothecin resistance. Because both mutation sites are near the catalytic active center, this observation raises the possibility that camptothecin may act at the vicinity of the catalytic active site of the enzyme-camptothecin-DNA complex.     

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.