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1.
Bulgarein, a fungal metabolite, induced mammalian topoisomerase I-mediated DNA cleavage in vitro. The cleavage activity of bulgarein was comparable to that of camptothecin at a drug concentration range of 0.025-approximately 5 microM. The DNA cleavage induced by bulgarein was suppressed at concentrations above 12.5 microM. Treatment of a reaction mixture containing bulgarein and topoisomerase I with elevated temperature (65 degrees C) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I-mediated DNA cleavage induced by bulgarein is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex." Intensity of cleaved DNA fragments induced by bulgarein with topoisomerase I was different from that induced by camptothecin. Bulgarein inhibited catalytic activities of both topoisomerase I and topoisomerase II. The changes in absorption spectra of bulgarein in the visible region observed upon addition of increasing amounts of calf thymus DNA indicate that bulgarein interacts with DNA. DNA (un)winding assay by two-dimensional gel electrophoresis showed that bulgarein induced the winding of DNA in the opposite direction to that of an intercalator so that positively supercoiled molecules are produced. Thus, bulgarein represents a new class of drugs which stabilizes the cleavable complex of topoisomerase I and alters the structure of DNA in a manner leading to a tightening of the helical twist. 相似文献
2.
E Kanematsu T Deguchi M Yasuda T Kawamura Y Nishino Y Kawada 《Canadian Metallurgical Quarterly》1998,42(2):433-435
The gyrA and parC genes of 31 clinical isolates of Enterococcus faecalis, including fluoroquinolone-resistant isolates, were partially sequenced and analyzed for target alterations. Topoisomerase IV may be a primary target in E. faecalis, but high-level fluoroquinolone resistance was associated with simultaneous alterations in both GyrA and ParC. 相似文献
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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. 相似文献
5.
Topoisomerase II is the cytotoxic target for a number of clinically relevant antitumor drugs. Berberrubine, a protoberberine alkaloid which exhibits antitumor activity in animal models, has been identified as a specific poison of topoisomerase II in vitro. Topoisomerase II-mediated DNA cleavage assays showed that berberrubine poisons the enzyme by stabilizing topoisomerase II-DNA cleavable complexes. Subsequent proteinase K treatments revealed that berberrubine-induced DNA cleavage was generated solely by topoisomerase II. Topoisomerase II-mediated DNA religation with elevated temperature revealed a substantial reduction in DNA cleavage induced by berberrubine, to the extent comparable to that of other prototypical topoisomerase II poison, etoposide, suggesting that DNA cleavage involves stabilization of the reversible enzyme-DNA cleavable complex. However, the step at which berberrubine induces cleavable complex may differ from that of etoposide as revealed by the difference in the formation of the intermediate product, nicked DNA. This suggests that berberrubine's primary mode of linear formation may involve trapping nicked molecules, formed at transition from linear to covalently closed circular DNA. Unwinding of the duplex DNA by berberrubine is consistent with an intercalative binding mode for this compound. In addition to the ability to induce the cleavable complex mediated with topoisomerase II, berberrubine at high concentrations was shown to specifically inhibit topoisomerase II catalytic activity. Berberrubine, however, did not inhibit topoisomerase I at concentrations up to 240 microM. Cleavage sites induced by topoisomerase II in the presence of berberrubine and etoposide were mapped in DNA. Berberrubine induces DNA cleavage in a site-specific and concentration-dependent manner. Comparison of the cleavage pattern of berberrubine with that of etoposide revealed that they share many common sites of cleavage. Taken together, these results indicate that berberrubine represents a new class of antitumor agent which exhibits the topoisomerase II poison activity as well as catalytic inhibition activity and may have a potential clinical value in cancer treatment. 相似文献
6.
We have observed that in the absence of hydrogen peroxide the Fe(III)-bleomycin (BLM) complex exhibits high DNA cleavage efficiency, converting supercoiled Form I DNA (pBR322 or phix174) to Form II (nicked, relaxed circular); the present study may give an important clue to elucidate the fact that iron-bleomycin mediated double-strand DNA cleavage requires at least one molecule of oxygen (O2) over the amount required to form 'activated bleomycin." 相似文献
7.
G Capranico M Binaschi ME Borgnetto F Zunino M Palumbo 《Canadian Metallurgical Quarterly》1997,18(9):323-329
Chemical agents able to interfere with DNA topoisomerases are widespread in nature, and some of them have outstanding therapeutic efficacy in human cancer and infectious diseases. DNA topoisomerases are essential enzymes that govern DNA topology during fundamental nuclear metabolic processes. Topoisomerase-interfering compounds can be divided into two general categories based on the mechanism of drug action: poisons and catalytic inhibitors. In past years, investigations of the DNA sequence selectivity of topoisomerase II poisons have identified structural and molecular determinants of drug activity, and indicated that the drug receptor is likely to be at the protein-DNA interface. Moreover, the available results indicate that the biologically relevant DNA-binding activity of topoisomerase poisons is basically protein-mediated and this is discussed in this issue by Giovanni Capranico and colleagues. This suggests that topoisomerase poisons may represent a useful paradigm for small compounds able to bind to protein-DNA interfaces in a site-selective manner, thus increasing the affinity of DNA-binding proteins for specific genomic sites. 相似文献
8.
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. 相似文献
9.
Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5. 相似文献
10.
The pattern of sites for cleavage mediated by topoisomerase II was determined in 830 kb of cloned DNA from the Drosophila X chromosome, with the objectives of comparing it with mapped structural and functional landmarks and examining if the correlations with such landmarks reported in individual loci can be generalized to a region approximately 100 times longer. The relative frequencies of topoisomerase II cleavage sites in 247 restriction fragments from 67 clones were quantified by hybridization with probes prepared from DNA fragments which abutted all cleavage sites in each clone, selected through the covalently bound topoisomerase II subunit; the specificity and quantitative nature of this method were demonstrated using a plasmid DNA model. The 12 restriction fragments with strong nuclear scaffold attachment (SAR) activity, of which seven possess autonomous replication (ARS) activity, show statistically strong coincidence or contiguity ( P =0.11) with regions of high topoisomerase II cleavage site frequency. These regions show no correlation with repetitive sequence or A/T or C/G content and some extend over >10 kb; their sensitivity is therefore unlikely to be due to alternating purine-pyrimidine repeats or regions of Z conformation, which are preferred motifs. The hypothesis that they possess intrinsic curvature is consistent with the similarity of their length and spacing to regions of predicted curvature in the 315 kb DNA of Saccharomyces cerevisiae chromosome III and with the reported strong binding preference of topoisomerase II for curved DNA. The topoisomerase II cleavage pattern in this DNA further shows that its relationships to functional properties seen in individual loci, especially to MAR/SAR and ARS activity and to the restricted accessibility of DNA to topoisomerase II in vivo, can be generalized to much longer regions of the genome. 相似文献
11.
Alterations in RNA polymerase structure can be detected using initial trypsin cleavage rates as a conformational probe. Both template (poly[d(A-T) . d(A-T)] and the RNA polymerase inhibitor, heparin, alter the rates at which the subunits of the enzyme are cleaved. However, while the presence of poly[d(A-T) . d(A-T)] slows the cleavage of subunits beta, sigma, and alpha by trypsin, heparin accelerates the cleavage of beta and sigma. Furthermore, the presence of heparin does not prevent the effect of poly[d(A-T) . d(A-T)] on the beta and sigma cleavage rates. Thus, heparin does not eliminate the interaction between DNA and RNA polymerase. That heparin does alter the nature of this interaction is demonstrated by the fact that template decreases the trypsin cleavage rate of subunit alpha in the absence, but not in the presence, of heparin. Like heparin, the addition of RNA to the reaction increases the accessibility of beta and sigma to trypsin. Hence the interaction of heparin with RNA polymerase may mimic the product, rather than the template, interaction. 相似文献
12.
A partial DNA duplex containing a high efficiency topoisomerase I cleavage site was substituted singly at each of three sites with 3'-deoxyadenosine. Depending on the site of substitution, the facility of the topoisomerase I-mediated cleavage or ligation reactions was altered. Inclusion of the modified nucleoside at the 5'-end of the acceptor oligonucleotide diminished the rate of religation following substrate cleavage by the enzyme. 相似文献
13.
gyrA and parC mutations have been identified inn Streptococcus pneumoniae mutants stepwise selected for resistance to sparfloxacin, an antipneumococcal fluoroquinolone. GyrA mutations (at the position equivalent to resistance hot spot Ser-83 in Escherichia coli GyrA) were found in all 17 first-step mutants examined and preceded DNA topoisomerase IV parC mutations (at Ser-79 or Glu-83), which appeared only in second-step mutants. The targeting of gyrase by sparfloxacin in S. pneumoniae but of topoisomerase IV by ciprofloxacin indicates that target preference can be altered by changes in quinolone structure. 相似文献
14.
During the period from November 1972 to February 1975, 39 patients received second renal grafts in our institution. The clinical course of the patients was analyzed and compared with 121 patients who received only one graft during the same period. The graft survival either from living related or cadaveric sources was inferior in the second graft group. However, mortality was not increased by re-transplantation. Major differences were noted in the occurrence of hyperacute or accelerated type rejections. There was a high incidence of this type of rejection in the second graft group, especially in the simultaneous retransplant group. 相似文献
15.
DA Burden PS Kingma SJ Froelich-Ammon MA Bjornsti MW Patchan RB Thompson N Osheroff 《Canadian Metallurgical Quarterly》1996,271(46):29238-29244
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. 相似文献
16.
The aim of the study was the definition of the clinical features and survival of 27 resected cases of distal bile duct carcinoma. This neoplasm accounted for 14% of all periampullary malignancies treated by pancreaticoduodenectomy between 1990 and 1996. Jaundice was present in 96% of patients, but was the first symptom only in 78%. Preoperative investigations allowed to recognize distal bile duct cancer in a minority of patients (41%). Operative mortality and morbidity were 3.7 and 44%, respectively. Most patients (88%) were assigned to UICC stage IV-A. Postoperative survival was not significantly better than survival of 101 patients undergoing pancreaticoduodenectomy for pancreatic ductal carcinoma; median survival was 22 months, with a 13% 5-year survival rate. Determinants of a better prognosis were UICC stage 相似文献
17.
Z Li T Deguchi M Yasuda T Kawamura E Kanematsu Y Nishino S Ishihara Y Kawada 《Canadian Metallurgical Quarterly》1998,42(12):3293-3295
We examined 22 clinical isolates of Staphylococcus epidermidis to analyze the association of alterations in GyrA and ParC with fluoroquinolone resistance. The simultaneous presence of GyrA and ParC alterations was associated with a high level of fluoroquinolone resistance in the clinical isolates of S. epidermidis. 相似文献
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BC Baguley F Leteurtre JF Riou GJ Finlay Y Pommier 《Canadian Metallurgical Quarterly》1997,33(2):272-279
AMCA (methyl N-[4-(9-acridinylamino)-2-methoxyphenyl]carbamate hydrochloride), an amsacrine analogue containing a methylcarbamate rather than a methylsulphonamide side chain, contrasts with amsacrine, doxorubicin and etoposide in its relatively high cytotoxicity against non-cycling tumour cells. AMCA bound DNA more tightly than amsacrine, but the DNA base selectivity of binding, as measured by ethidium displacement from poly[dA-dT].[dA-dT] and poly[dG-dC].[dG-dC], was unchanged. AMCA-induced topoisomerase cleavage sites on pBR322, C-MYC and SV40 DNA were investigated using agarose or sequencing gels. DNA fragments were end-labelled, incubated with purified topoisomerase II from different mammalian sources and analysed after treatment with sodium dodecylsulphate/proteinase K. AMCA stimulated the cleavage activity of topoisomerase II, but the DNA sequence selectivity of cleavage was different from that of amsacrine and other topoisomerase inhibitors. It was similar to that of the methoxy derivative of AMCA, indicating that the changed specificity resulted from the carbamate group rather than from the methoxy group. The pattern of DNA cleavage induced by AMCA was similar for topoisomerase II alpha and II beta. 相似文献