Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

We have compared topoisomerase I and II cleavage sites on the actin 5C and 57A genes and the hsp70 genes in Drosophila Kc cells using the inhibitors camptothecin (topoisomerase I specific) and VM-26 (topoisomerase II specific) to assess the role of these enzymes in transcriptional regulation. Topoisomerase I cleavage sites were localized to the transcribed regions of the actin 5C and hsp70 genes and were present only when these genes were active. The actin 57A gene, shown previously to be inactive in Kc cells, had no detectable topoisomerase I cleavage sites. In contrast to topoisomerase I, topoisomerase II cleavage sites could be detected on transcriptionally active and inactive actin and hsp70 DNA sequences. Topoisomerase II cleavage sites on the inactive hsp70 gene were primarily localized to the very 5' end of the transcribed region of the gene. However, upon heat-induced activation of hsp70 transcription, topoisomerase II cleavage rapidly shifted from the 5' to the 3' end of the gene. Then, during the shutdown of hsp70 expression, there was a gradual reappearance of topoisomerase II cleavage at the 5' end of the gene that temporally correlated with the repression of hsp70 transcription. There was a similar preferential association of topoisomerase II with the 5' ends of transcriptionally repressed actin 5C and 57A genes. These results demonstrate that there are marked differences in how topoisomerases I and II interact with transcriptionally active and inactive regions of chromatin. In addition, we have identified an unusual type of topoisomerase II binding site that is preferentially associated with the 5' ends of inactive hsp70 and actin genes, suggesting that this enzyme may facilitate changes in chromatin structure that are associated with repression of gene transcription.
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PMID:Analysis of topoisomerase I and II cleavage sites on the Drosophila actin and Hsp70 heat shock genes. 131 49

In vitro transcription was reconstituted with HeLa cell transcription factors and RNA polymerase II, which were essentially free from DNA topoisomerase activities. DNA templates with defined negative superhelical densities were tested for transcription activity. Transcription of the Bombyx mori fibroin gene increases and plateaus from templates of increasing superhelicity, and transcription from the adenovirus 2 major late promoter rises and then falls, while transcription of the Drosophila hsp70 gene remains unchanged. Dissection of transcription into pre and post-initiation steps by the use of Sarkosyl reveals that formation of a preinitiation complex on the fibroin gene or the adenovirus 2 major late promoter is slow on relaxed DNA and accelerated by DNA superhelicity. On the contrary, the preinitiation complex assembles rapidly on the hsp70 gene irrespective of DNA topology. As is the case with the fibroin gene promoter, DNA superhelicity appears to facilitate the interaction of transcription factor IID to the adenovirus 2 major late promoter.
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PMID:DNA superhelicity affects the formation of transcription preinitiation complex on eukaryotic genes differently. 164 22

In a posterior silk gland extract, covalently closed circular (ccc) DNA is in a superhelical state that supports more transcription of fibroin gene than does linear DNA. A HeLa cell extract showed neither the supercoiling activity nor the preference for the transcription of ccc DNA over linear DNA. These activities could be added to the HeLa cell extract. Phosphocellulose fractionation of the posterior silk gland extract yielded a flow-through fraction and a 0.6 M KCl eluate fraction that were required for the supercoiling and for the efficient transcription of the ccc template in the acceptor HeLa cell extract. The 0.6 M KCl fraction had a DNA topoisomerase II activity, and the flow-through fraction contained a supercoiling factor that, with the aid of topoisomerase II, introduced negative supercoils into ccc DNA. When both fractions were added to the posterior silk gland extract, more supercoiling occurred than with the extract alone. Several genes were optimally transcribed under various extents of supercoiling. The fibroin gene and adenovirus 2 major late promoter were fully transcribed as partially supercoiled templates. The sericin gene required more supercoiling for full transcription, whereas no preference for supercoiling was seen with the transcription of hsp70. These results suggest that DNA topology plays a role in the regulation of gene expression.
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PMID:In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling. 282

Similar to its inhibitory effect on mammalian DNA topoisomerase II, the cytotoxic drug VM26 (teniposide) also interferes with the breakage-reunion reaction of Drosophila melanogaster DNA topoisomerase II. VM26 induces topoisomerase II-mediated DNA breakage in vitro and in cultured D. melanogaster cells presumably by stabilizing an enzyme-DNA cleavable complex. The drug-induced DNA breaks on D. melanogaster hsp70 genes were mapped in cultured cells using the indirect end-labeling procedure. Multiple and specific cleavage sites occurred at both the 3' and 5' ends of the hsp70 genes. A number of these cellular topoisomerase II cleavage sites mapped close to the DNase I-hypersensitive regions of the hsp70 genes. The intensities of several topoisomerase II cleavage sites changed significantly on heat shock induction. Treatment of cultured D. melanogaster cells with VM26 at 25 degrees C resulted in the stimulation of transcription of the hsp70 genes. These results suggest that inhibition of DNA topoisomerase II may lead to heat shock transcription.
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PMID:In vivo localization of DNA topoisomerase II cleavage sites on Drosophila heat shock chromatin. 302 86

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.
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PMID:Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding. 303 51

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.
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PMID:The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery. 824 70

Previous in vivo studies have identified a prominent 4'-demethylepipodophyllotoxin-9-(4,6-O-thionylidine-beta-D-g lucopyranoside) (VM-26)-induced double-stranded topoisomerase II cleavage site at approximately +80 relative to the start of Drosophila hsp70 transcription (Kroeger, P. E., and Rowe, T. C. (1992) Biochemistry 31, 2492-2502). Topoisomerase II binding at this site correlated with the repression of hsp70 transcription suggesting that this protein-DNA interaction was important in the regulation of hsp70 gene expression. In this paper, we investigated the interaction of purified Drosophila topoisomerase II with a 271-base pair DNA fragment containing the +80 region of the hsp70 gene using the topoisomerase II-specific inhibitor VM-26. VM-26-induced topoisomerase II cleavage of the hsp70 DNA resulted in a major 4-base staggered double-stranded break at +84. In the absence of ATP the +84 site was the only significant VM-26-induced cleavage site. Addition of ATP to the reaction resulted in a stimulation of topoisomerase cleavage throughout the 271-base pair DNA fragment. Deletion analyses determined that approximately 15 to 25 bp of flanking sequence were required for efficient cleavage at most topoisomerase II sites within the hsp70 DNA. However, in the case of the +84 site, topoisomerase cleavage still occurred even when this site was split in half by the restriction enzyme PstI. Topoisomerase II cleavage of both "half-site" DNA molecules occurred at the correct positions on the 4-base single-stranded DNA overhangs generated by PstI. Cleavage was reversible indicating that topoisomerase II could reseal the single-stranded DNA break formed in each half-site substrate. Denaturation of the half-site molecules abolished topoisomerase II cleavage suggesting that cleavage required the duplex region adjacent to the single-stranded cleavage site. Identification of this unusual half-site substrate provides additional evidence that double-stranded cleavage of DNA by topoisomerase II occurs via two sequential single-stranded breaks.
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PMID:Analysis of topoisomerase II-mediated DNA cleavage in the 5'-region of the Drosophila hsp70 gene. Identification of a novel half-site DNA substrate for topoisomerase II cleavage. 839 63

Apoptosis is a well-established cellular mechanism for selective cell deletion during development. However, little is known about the expression of an apoptotic pathway and its role in determining the relative sensitivity of the early, pre-gastrula, avian embryo to stress-induced cell death. We examined the sensitivity of avian blastodermal cells to engage in apoptosis upon exposure to etoposide, a topoisomerase II-inhibitor that rapidly and efficiently induces apoptosis in many cell types. We found that while the blastodermal cells are capable of engaging in apoptosis, they are highly resistant to such induction with respect to both concentration of drug required and length of exposure, even when compared to a tumor cell line with a known multi-drug resistant phenotype. Additionally, we assessed the expression of several candidate regulatory genes in blastodiscs from infertile eggs (i.e., maternal RNA transcripts), blastodermal cells immediately following oviposition, and various stages of early development up to gastrulation. This analysis revealed that several genes whose products have anti-apoptotic activity, including bcl-2, bcl-xL, hsp70, grp78 and the glutathione S-transferases, are expressed as early as the stage 1 embryo in the newly oviposited egg. These transcripts are also found in the infertile blastodisc, suggesting a role for maternally derived transcripts in the protection of the oocyte and zygote. Significantly, constitutive levels of hsp70 mRNA exceeded those of the other anti-apoptotic genes in the blastodermal cells. These results contribute to an emerging picture of stress resistance at the earliest stages of avian embryo development which involves multiple anti-apoptotic genes that act at different regulatory points in the apoptotic cascade.
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PMID:Expression of cell death regulatory genes and limited apoptosis induction in avian blastodermal cells. 974 Mar 20