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 previously shown that a cloned 480 bp DNA fragment that spans the 3'-enhancer region of the avian beta-globin gene cluster can become very tightly, perhaps covalently, bound to protein in avian nuclear matrices in vitro [Zenk et al. (1990) Biochemistry 29, 5221-5226]. This binding was not tissue-specific and was probably not mediated by topoisomerase enzymes. In the present study, we have examined avian nuclear matrices (or scaffolds) for the presence of very tight cellular DNA-protein complexes in the region of the beta-globin gene enhancer and of several other avian genes. Nuclear matrices were prepared by both high- and low-salt methods, and protein-DNA complexes were isolated by SDS/K+ precipitation after restriction enzyme digestion. In adult reticulocytes, up to 30% of the intact 3800 bp HindIII-EcoRI fragment that encompasses the beta-globin enhancer element may be very tightly bound to nuclear matrix protein. In adult avian thymus nuclei, the beta-globin enhancer is neither matrix-associated nor tightly bound to protein. In contrast, a 5.0-kb HindIII fragment of the malic enzyme gene is very tightly bound to nuclear matrix-associated protein in thymus cells, but not reticulocytes. The malic enzyme gene is active in thymus cells, and not in reticulocytes. These results suggests that certain regions of cellular DNA are very tightly, perhaps covalently, attached to nuclear matrix-associated proteins. Attachment follows a tissue-specific pattern that is associated with transcriptional activity.
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PMID:Avian nuclear matrix proteins bind very tightly to cellular DNA of the beta-globin gene enhancer in a tissue-specific fashion. 204 26

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken beta-globin locus, which contains four globin genes (5'-rho-beta H-beta A-epsilon-3'). In the 65 kilobases (kb) mapped, 12 strong hypersensitive sites were found clustered within the 25-kb region from 10 kb upstream of rho to just downstream of epsilon. The strong sites were grouped into several classes based on their tissue distribution, developmental pattern, and location. (i) One site was present in all cells examined, both erythroid and nonerythroid. (ii) Three sites, located upstream of the rho-globin gene, were present at every stage of erythroid development, but were absent from nonerythroid cells. (iii) Four sites at the 5' ends of each of the four globin genes were hypersensitive only in the subset of erythroid cells that were transcribing or had recently transcribed the associated gene. (iv) Another three sites, whose pattern of hypersensitivity also correlated with expression of the associated gene, were found 3' of rho, beta H, and epsilon. (v) A site 3' of beta A and 5' of epsilon was erythroid cell specific and present at all developmental stages, presumably reflecting the activity of this enhancer throughout erythroid development. We also mapped the topo II sites in this locus, as determined by teniposide-induced DNA cleavage. All strong teniposide-induced cleavages occurred at DNase I-hypersensitive sites, while lesser amounts of cleavage were observed in transcribed regions of DNA. Most but not all of the DNase I-hypersensitive sites were topo II sites. These data are consistent with the hypothesis that, in vivo, topo II preferentially acts on nucleosome-free regions of DNA but suggest that additional topo II regulatory mechanisms must exist.
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PMID:Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus. 216 May 85

Current evidence suggests that DNA is covalently attached to proteins in the nuclear matrix of eukaryotic cells and that specific DNA sequences are tightly associated with the nuclear matrix. However, it has not been documented that specific DNA sequences can become covalently attached to nuclear matrix protein. We have examined the binding of cloned DNA sequences that contain the avian beta-globin gene enhancer, a region previously shown to be matrix associated in erythroid cells in vivo, with nuclear matrices from several avian tissue sources to determine if covalent DNA-protein bonds are formed. Our results indicate that sequence-specific DNA-protein complexes that are resistant to denaturation by SDS, boiling, and phenol and disulfide reduction are formed. Excess protein, capable of forming very tight bonds with DNA that contains the beta-globin gene enhancer, is present in cells in which matrix attachment of this DNA sequence is not detected in vivo. Evidence is presented that suggests that the protein to which DNA forms very tight bonds is not topoisomerase II. These results are discussed in relation to current models of the nuclear matrix and the utility of in vitro assays of matrix attachment regions using cloned DNA.
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PMID:A nuclear matrix protein binds very tightly to DNA in the avian beta-globin gene enhancer. 238 42

One postulated mechanism for how the SV40 enhancer stimulates transcription of linked genes involves the enhancer as a binding site for a sequence-specific "gyrase" activity. We sought to test this hypothesis directly by constructing a novel heteroduplex circle, termed a tailed-circle, in which one of the strands contains an extra palindromic sequence base-paired into a hairpin structure. The human beta-globin gene is placed in the circle and the SV40 enhancer on the hairpin tail, where a bound topoisomerase cannot supercoil the circle. Upon transfection of this DNA into HeLa cells the SV40 enhancer on the hairpin arm is still able to stimulate transcription of the beta-globin gene. Southern blot analysis of the DNA after transfection does not demonstrate any repair or replication of the tailed-circle in vivo. These results argue against the sequence-specific gyrase model for SV40 enhancer action.
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PMID:Transcription of the human beta-globin gene is stimulated by an SV40 enhancer to which it is physically linked but topologically uncoupled. 301 Dec 74

We have identified a region of the beta-globin gene that is attached constitutively to histone-depleted murine erythroleukemia cell nuclei. This region spans 800 bp and is located at -300 to -1100 bp upstream from the site of transcriptional initiation. Attachment is not altered by transcriptional activation of the beta-globin gene during induction to terminal differentiation, and the same region of the beta-globin gene is attached to histone-depleted myeloma cell nuclei (NS-1). The attached region contains an A/T-rich section, in addition to a sequence closely related to the Drosophila topoisomerase II consensus cleavage sequence. No comparable site of attachment of the alpha 1-globin gene was detected when a region spanning 1.5 kb 5' to 0.5 kb 3' of the region of transcription was studied.
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PMID:Constitutive attachment of murine erythroleukemia cell histone-depleted DNA loops to nuclear scaffolding is found in the beta-major but not the alpha 1-globin gene. 322 84

Active genes are known to have an altered chromatin structure that is preferentially sensitive to digestion with DNAase I. We find that when chicken red blood cells are incubated in media containing the topoisomerase II inhibitor novobiocin, the preferential DNAase I sensitivity of the active beta-globin genes is reversed in vivo with as little as 20 min of drug treatment. Control experiments suggest that inhibition of a topoisomerase II is responsible for this alteration in active gene conformation. Reversal of DNAase I sensitivity can also be induced in vitro by partial cleavage of the nuclear DNA with staphylococcal nuclease. We propose that the altered structure around active genes is maintained by continuous DNA supercoiling and that in the absence of this superhelical tension active chromatin reverts to a less DNAase I-sensitive ground state.
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PMID:Torsional stress promotes the DNAase I sensitivity of active genes. 609 6

In a search for factors that influence the process of erythroid differentiation at the molecular level, we have identified UB2, a nuclear protein factor that was originally observed for its ability to bind to a very specific and highly conserved sequence motif present in human, mouse, rabbit, and chicken beta-globin genes, as well as carbonic anhydrase I, c-myb, and the immunoglobulin heavy chain enhancer region. It was also observed for its appearance in undifferentiated but not differentiated mouse erythroleukemia cells. Purification of UB2 by DEAE-cellulose chromatography and repeated passages through a DNA affinity column, revealed a complex pattern with three major components of 170, 116, and 48 kDa, respectively. The 170-kDa protein was identified as topoisomerase (topo) II by Western blot analysis, catalytic assays, and antibody interference with UB2 binding. The complex topo II in UB2, however, has a more stringent sequence requirement for DNA binding than does topo II. The 116-kDa protein has been determined to be a proteolytic product of topo II. The chromosome scaffold protein 2 (135 kDa) copurified with UB2, and anti-scaffold protein 2 serum inhibited UB2 binding to DNA.
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PMID:Purification and characterization of a nuclear DNA-binding factor complex containing topoisomerase II and chromosome scaffold protein 2. 838 2

The effect of VM-26, a topoisomerase II targeting drug, on IW32 murine erythroleukemia cells was investigated. The VM-26 induced IW32 cells to differentiate at a non-toxic but cytostatic concentration (0.01 microgram/ml). More than 40% of the cells were induced to synthesize hemoglobin, and cells were arrested in G2/M phase of the cell cycle. Levels of beta-globin mRNA also increased significantly. Cells became committed to erythroid maturation after 16 h of continuous drug exposure. Replacement with fresh VM-26 after 48 h of drug treatment further increased the hemoglobin containing cells to greater than 80%. Unlike other drug induced erythroleukemia cell differentiation, c-myc mRNA expression was not affected by VM-26. Inhibition of topoisomerase II activity was observed during the first 12 h of VM-26 treatment; however, elevated enzyme activity was found thereafter. Northern blot analysis showed significant increase in the expression of topoisomerase IIalpha mRNA at 12 and 24 h after VM-26 addition. These findings indicate that VM-26 inhibited the activity of topoisomerase II and promoted the committed differentiation of IW32 cells along the erythroid pathway. In addition, a parallel increase in mRNA and activity levels of topoisomerase II in differentiated cells suggests that regulation of the enzyme expression occurred in the VM-26 induced erythroid maturation.
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PMID:Regulation of topoisomerase II expression during the VM-26 induced differentiation of IW32 murine erythroleukemia cells. 863 20

A 52 base pair alternating purine-pyrimidine (RY) repeat sequence lies in the 5' upstream region of the human beta-globin gene. The structural transition of a plasmid containing this repeat was analyzed by two-dimensional gel electrophoresis. These conformational studies indicate that the 52 bp RY repeat undergoes local transition from the right-handed B-DNA into a cruciform DNA under torsional stress and the transition initiates at a threshold level of negative supercoiling (-sigma = 0.042). The superhelicity-dependent S1 nuclease cleavage sites were mapped only within the RY repeat and no nicking was observed outside of the repeat. In view of the fact that DNA topoisomerase II is highly reactive towards RY repeat which can adopt unusual DNA conformation, we have investigated the effects of the superhelicity-dependent conformational transition of the 52 bp RY repeat on topoisomerase II cleavages. Cleavage reactions were performed on the pRYG plasmid with varying levels of negative superhelical densities ranging from 0 to -0.074. Under the low torsional stress, topoisomerase II cleavage activity at the RY repeat gradually increased with the increasing levels of negative superhelical densities. However, over a threshold level of negative supercoiling for cruciform conformation, the intensities of enzyme cleavage sites at the RY repeat were essentially identical. These results suggest that topoisomerase II can bind and cleave the cruciform structure in a dynamic process identical to duplex B-DNA.
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PMID:Topoisomerase II-mediated DNA cleavage on the cruciform structure formed within the 5'upstream region of the human beta-globin gene. 974 29

We present titrations of the human delta beta-globin gene region with DNA minor groove binders netropsin, bisnetropsin, distamycin, chromomycin and four bis-quaternary ammonium compounds in the presence of calf thymus topoisomerase II and DNase I. With increasing ligand concentration, stimulation and inhibition of enzyme activity were detected and quantitatively evaluated. Additionally we show a second type of stimulation, the appearance of strong new topoisomerase II cleavage sites at high ligand concentrations. The specific binding sites of the minor groove binders of the DNA sequence and their microscopic binding constants were determined from DNase I footprints. A binding mechanism for minor groove binders is proposed in order to explain these results especially when ligand concentration is increased.
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PMID:DNA binding properties of minor groove binders and their influence on the topoisomerase II cleavage reaction. 977 Jun 48


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