Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.3 (topoisomerase)
 9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Diospyrin is a plant product that has significant inhibitory effect on the growth of Leishmania donovani promastigotes. This compound inhibits the catalytic activity of DNA topoisomerase I of the parasite. Like camptothecin, it induces topoisomerase I mediated DNA cleavage in vitro. Treatment of DNA with diospyrin before addition of topoisomerase I has no effect. Preincubation of topoisomerase I with diospyrin before the addition of DNA in the relaxation reaction increases this inhibition. Our results suggest that this bis-naphthoquinone compound exerts its inhibitory effect by binding with the enzyme and stabilizing the topoisomerase I-DNA "cleavable complex." Diospyrin is a specific inhibitor of the parasitic topoisomerase I. It does not inhibit type II topoisomerase of L. donovani and requires much higher concentrations to inhibit type I topoisomerase of calf thymus. The potent inhibitory effect of diospyrin on type I DNA topoisomerase from L. donovani can be exploited for rational drug design in human leishmaniasis.
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PMID:Diospyrin, a bisnaphthoquinone: a novel inhibitor of type I DNA topoisomerase of Leishmania donovani. 985 27

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.
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PMID:Rhodobacter capsulatus DNA topoisomerase I purification and characterization. 991 36

In order to clone the gene encoding a type I DNA topoisomerase from Leishmania donovani, a PCR-amplified DNA fragment obtained with degenerate oligodeoxyribonucleotides was used to screen a genomic library from this parasite. An open reading frame of 1905 bases encoding a putative protein of 635 amino acid residues was isolated. A substantial part of the protein shares a significant degree of homology with the sequence of other known members of the IB topoisomerase family, in a highly conserved region of these enzymes termed the core domain. However, homology is completely lost after this conserved central core. Moreover, no conventional active tyrosine site could be identified. In fact, the protein expressed in Escherichia coli did not show any relaxation activity in vitro and was unable to complement a mutant deficient in topoisomerase I activity. The results of Southern blot experiments strongly suggested that the cloned gene was not a pseudogene. Northern analysis revealed that the gene was transcribed in its full length and also excluded the possibility that some form of splicing is necessary to produce a mature messenger. Furthermore, our results indicate that the gene is preferentially expressed in actively growing L.donovani promastigotes and that it is also expressed in other kinetoplastid parasites.
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PMID:Characterization of a Leishmania donovani gene encoding a protein that closely resembles a type IB topoisomerase. 1037 92

Vaccinia virus encodes a type I DNA topoisomerase that is highly conserved in all known poxviruses. Although the structure and catalytic activity of the enzyme were well studied, little was known about its biological function. The viral topoisomerase was thought to be essential, and roles in DNA replication, recombination, concatemer resolution, and transcription were suggested. Here, we demonstrated that the topoisomerase is not essential for replication of vaccinia virus in cultured cells, although deletion mutants formed fewer and smaller plaques on cell monolayers than wild-type virus. Purified mutant virus particles were able to bind and enter cells but exhibited reduced viral early transcription and a delay in DNA replication. Infecting with a high number of virus particles increased early mRNA and accelerated viral DNA synthesis. Processing of viral DNA concatemers into unit-length genomes was unimpaired at either a low or high multiplicity of infection. The data suggest that the primary, perhaps only, role of the poxvirus topoisomerase is to increase early transcription, which takes place within virus cores in the cytoplasm of infected cells. Because the topoisomerase functions early in infection, drugs capable of penetrating the virus core and irreversibly damaging DNA by trapping nicked DNA-topoisomerase intermediates could make potent antiviral agents.
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PMID:Poxvirus DNA topoisomerase knockout mutant exhibits decreased infectivity associated with reduced early transcription. 1297 42

Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover.
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PMID:Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay. 1555 7

DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.
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PMID:Purification and characterization of a novel ATP-independent type I DNA topoisomerase from a marine methylotroph. 1585 May 56

Most chemotherapy regimens contain at least one DNA-damaging agent that preferentially affects the growth of cancer cells. This strategy takes advantage of the differences in cell proliferation between normal and cancer cells. Chemotherapeutic drugs are usually designed to target rapid-dividing cells because sustained proliferation is a common feature of cancer [1,2]. Rapid DNA replication is essential for highly proliferative cells, thus blocking of DNA replication will create numerous mutations and/or chromosome rearrangements-ultimately triggering cell death [3]. Along these lines, DNA topoisomerase inhibitors are of great interest because they help to maintain strand breaks generated by topoisomerases during replication. In this article, we discuss the characteristics of topoisomerase (DNA) I (TOP1) and its inhibitors, as well as the underlying DNA repair pathways and the use of TOP1 inhibitors in cancer therapy.
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PMID:Inhibition of Topoisomerase (DNA) I (TOP1): DNA Damage Repair and Anticancer Therapy. 2628 59


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