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Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The TOP3 gene of the yeast Saccharomyces cerevisiae was postulated to encode a DNA topoisomerase, based on its sequence homology to Escherichia coli DNA topoisomerase I and the suppression of the poor growth phenotype of top3 mutants by the expression of the E. coli enzyme (Wallis, J.W., Chrebet, G., Brodsky, G., Golfe, M., and Rothstein, R. (1989) Cell 58, 409-419). We have purified the yeast TOP3 gene product to near homogeneity as a 74-kDA protein from yeast cells lacking DNA topoisomerase I and overexpressing a plasmid-borne TOP3 gene linked to a phosphate-regulated yeast PHO5 gene promoter. The purified protein possesses a distinct DNA topoisomerase activity: similar to E. coli DNA topoisomerases I and III, it partially relaxes negatively but not positively supercoiled DNA. Several experiments, including the use of a negatively supercoiled heteroduplex DNA containing a 29-nucleotide single-stranded loop, indicate that the activity has a strong preference for single-stranded DNA. A protein-DNA covalent complex in which the 74-kDa protein is linked to a 5' DNA phosphoryl group has been identified, and the nucleotide sequences of 30 sites of DNA-protein covalent complex formation have been determined. These sequences differ from those recognized by E. coli DNA topoisomerase I but resemble those recognized by E. coli DNA topoisomerase III. Based on these results, the yeast TOP3 gene product can formally be termed S. cerevisiae DNA topoisomerase III. Analysis of supercoiling of intracellular yeast plasmids in various DNA topoisomerase mutants indicates that yeast DNA topoisomerase III has at most a weak activity in relaxing negatively supercoiled double-stranded DNA in vivo, in accordance with the characteristics of the purified enzyme.
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PMID:Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase. 132 25

The nucleic acid-binding domain of Escherichia coli DNA topoisomerase III (Topo III) has been identified using a selection procedure designed to isolate inactive Topo III polypeptides. Deletion of this binding domain, contained in the carboxyl terminus of Topo III, results in a drastic reduction in the ability of the enzyme to bind to single-stranded DNA and RNA substrates. Successive truncation of the enzyme within this region results in the gradual loss of nucleic acid binding activity and in a gradual change in the mechanism of Topo III-catalyzed relaxation of negatively supercoiled DNA. The reduction of nucleic acid binding activity of the truncated polypeptides does not result in a loss of cleavage site specificity for the enzyme, suggesting that other amino acids are involved in the positioning of the nucleic acid within the nicking/closing site of the topoisomerase.
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PMID:The carboxyl-terminal residues of Escherichia coli DNA topoisomerase III are involved in substrate binding. 765 74

The binding of DNA topoisomerase III (Topo III) to a single-stranded DNA substrate containing a strong cleavage site has been examined. The minimal substrate requirement for Topo III-catalyzed cleavage has been determined to consist of 7 bases; 6 bases 5' to the cleavage site and only 1 base 3' to the site. Nuclease P1 protection experiments indicate that the enzyme also binds to its substrate asymmetrically, protecting approximately 12 bases 5' to the cleavage site and only 2 bases 3' to the cleavage site. A catalytically inactive mutant of Topo III shows the same protection pattern as the active polypeptide, indicating that Topo III is a site-specific binding protein as well as a topoisomerase. Consistent with this view, an oligonucleotide containing a cleavage site is a more effective inhibitor and is bound more efficiently by Topo III than an oligonucleotide without a cleavage site.
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PMID:Escherichia coli DNA topoisomerase III is a site-specific DNA binding protein that binds asymmetrically to its cleavage site. 755 40

The yeast TOP3 gene, encoding DNA topoisomerase III, and EST1 gene, encoding a putative telomerase, are shown to be abutted head-to-head on chromosome XII, with the two initiation codons separated by 258 bp. This arrangement suggests that the two genes might share common upstream regulatory sequences and that their products might be functionally related. A comparison of isogenic pairs of yeast TOP3+ and delta top3 strains indicates that the G1-3T repetitive sequence tracks in delta top3 cells are significantly shortened, by about 150 bp. Cells lacking topoisomerase III also show a much higher sequence fluidity in the subtelomeric regions. In delta top3 cells, clusters of two or more copies of tandemly arranged Y' elements have a high tendency of disappearing due to the loss or dispersion of the elements; similarly, a URA3 marker embedded in a Y' element close to the chromosomal tip shows a much higher rate of being lost relative to that in TOP3+ cells. These results suggest that yeast DNA topoisomerase III might affect telomere stability, and plausible mechanisms are discussed.
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PMID:Effects of yeast DNA topoisomerase III on telomere structure. 770 2

A synthetic strand of RNA has been designed so that it can adopt two different topological states (a circle and a trefoil knot) when ligated into a cyclic molecule. The RNA knot and circle have been characterized by their behavior in gel electrophoresis and sedimentation experiments. This system allows one to assay for the existence of an RNA topoisomerase, because the two RNA molecules can be inter-converted only by a strand passage event. We find that the interconversion of these two species can be catalyzed by Escherichia coli DNA topoisomerase III, indicating that this enzyme can act as an RNA topoisomerase. The conversion of circles to knots is accompanied by a small amount of RNA catenane generation. These findings suggest that strand passage must be considered a potential component of the folding and modification of RNA structures.
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PMID:An RNA topoisomerase. 879 Mar 55

The polypeptide encoded by the plasmid RP4 traE gene shows extensive protein sequence similarity to Escherichia coli topB, the gene encoding DNA topoisomerase III (Topo III). The traE gene product has been cloned into a bacteriophage T7-based transient expression system, and the polypeptide has been expressed and purified. The TraE protein exhibits topoisomerase activity similar to that of Topo III. Relaxation is stimulated by high temperature and low concentrations of Mg2+. In addition, similar to E. coli Topo III, the TraE protein is a potent decatenase and can substitute for Topo III activity in vivo. The biochemical properties of the TraE protein in vitro suggest that the protein may be involved in the resolution of plasmid DNA replication intermediates either during vegetative replication or in conjugative DNA transfer. Putative homologues of Topo III have been found to be encoded by other broad host range, conjugative plasmids isolated from both Gram-negative and Gram-positive organisms, suggesting that Topo III-like polypeptides may have an essential role in the propagation of many promiscuous plasmids.
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PMID:The traE gene of plasmid RP4 encodes a homologue of Escherichia coli DNA topoisomerase III. 923 64

The human DNA topoisomerase III (hTOP3) gene encodes a topoisomerase homologous to the Escherichia coli DNA topoisomerase I subfamily. To understand the mechanisms responsible for regulating hTOP3 expression, we have cloned the 5'-flanking region of the gene coding for the hTOP3 and analyzed its promoter activity. The presence of a single transcription initiation site was suggested by primer extension analysis. The hTOP3 gene promoter is moderately high in GC content and lacks a canonical TATA box, suggesting that hTOP3 promoter has overall similarity to promoters of a number of housekeeping genes. Examination of the promoter sequence indicated the presence of four Sp-1 consensus binding sequences and a putative initiator element surrounding the transcription initiation site. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the 52-base pair region from -326 to -275 upstream of the transcription initiation site includes a positive cis-acting element(s) for the efficient expression of hTOP3 gene. On the basis of gel mobility shift and supershift assays, we demonstrated that both YY1 and USF1 transcription factors can bind to the 52-base pair region. When HeLa cells were transiently transfected with a mutant construct which had disabled both YY1- and USF1-binding sites, the luciferase activity was greatly reduced, suggesting that these binding elements play a functional role in the basal activation of the hTOP3 promoter. Transfection studies with mutations that selectively impaired YY1 or USF1 binding suggested that both YY1 and USF1 function as activators in the hTOP3 promoter.
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PMID:Cloning and characterization of the 5'-flanking region for the human topoisomerase III gene. 974 94

We cloned cDNA encoding a novel mouse homologue of DNA topoisomerase III (mTOP3beta). The nucleotide sequence contains an open reading frame of 863 amino acids, and the deduced molecular mass of the coded protein is 96.9 kDa. The overall sequence of mTOP3beta has a 48 and 36% identity with mouse TOP3alpha at the nucleotide and amino acid level, respectively. DNA topoisomerase IIIbeta was expressed using a baculovirus expression system and purified. The purified DNA topoisomerase IIIbeta had activity to relax negatively supercoiled DNA. Relaxation of supercoiled DNA was partial at 37 degreesC and complete relaxation was observed at higher temperatures. mTOP3beta mRNA was strongly expressed in the testis and relatively strongly in the brain. The levels of TOP3beta mRNA in the testis increased slightly 14 days and considerably 17 days after birth, when the cells in the pachytene phase begin to appear and increase.
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PMID:Cloning of cDNA encoding a novel mouse DNA topoisomerase III (Topo IIIbeta) possessing negatively supercoiled DNA relaxing activity, whose message is highly expressed in the testis. 978 43

Eukaryotic DNA topoisomerase III was first identified by studying the hyper-recombination and slow growth phenotypes of yeast mutants. Topoisomerase III interacts with DNA helicase SGS1 and the two proteins are involved in DNA recombination, cellular aging and maintenance of genome stability. A human homolog of topoisomerase III has previously been identified. Here we report the identification of cDNAs and the determination of gene structure for a second human topoisomerase III gene. This novel gene expresses three alternatively spliced transcripts, which encode gene products different in the putative DNA-binding C-termini. The largest gene product of the novel topoisomerase III was expressed and shown to interact with SGS1 protein and partially rescue the slow growth defect of a yeast topoisomerase III mutant. The presence of more than one human topoisomerase III is reminiscent of mammalian topoisomerase II, which has two genetically distinct isoforms with different expression patterns and probably different functions in mammalian cells.
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PMID:A new human topoisomerase III that interacts with SGS1 protein. 992 31

One major function of DNA topoisomerase I in Escherichia coli is to repress R-loop formation during transcription elongation, which may otherwise inhibit cell growth. We have previously shown that the growth problems of topA mutants can be corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R-loop. The goal of the present study was to identify other potential regulators of R-loop formation. To this end, we have screened for multicopy suppressors of topA null mutations. As expected using this procedure, we cloned the rnhA gene encoding RNase H. In addition, we also identified the topB gene encoding DNA topoisomerase III as an efficient suppressor of topA null mutations and, hence, of R-loop formation. We show that DNA topoisomerase III is able to relax transcription-induced negative supercoiling both in vitro and in vivo. An R-loop is also shown to be a hot-spot for relaxation by DNA topoisomerase III, and we found that R-loop-dependent hypernegative supercoiling can be prevented by the activity of this topoisomerase in vivo. It is also shown that the topB gene can act synergistically with the rnhA gene to correct the growth defect of topA null mutants efficiently. This synergistic effect can be explained by the fact that some R-loops must not be degraded in order for the RNA to be available for protein synthesis. Topoisomerase III can presumably repress the formation of such R-loops or cause their destabilization to prevent RNA degradation. This is supported by the fact that overproduction of this topoisomerase corrects the negative effect of overexpressing RNase H activity on the growth of topA null mutants at low temperatures. Moreover, the fact that DNA topoisomerase III does not relax global supercoiling supports our previous conclusion that R-loop formation, and therefore the essential function of DNA topoisomerase I, involves local, rather than global, supercoiling.
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PMID:Isolation of the topB gene encoding DNA topoisomerase III as a multicopy suppressor of topA null mutations in Escherichia coli. 1063 77


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