Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Simian virus 40 (SV40) chromatin extracted from nuclei of infected monkey cells (CV1) was sedimented in neutral sucrose gradients, before and after digestion with bovine pancreatic RNase I-A or DNase I. DNA topoisomerase (TI) activity was found associated with RNase-resistant, DNase-sensitive SV40 nucleoprotein complexes. After polyacrylamide gel electrophoresis, a number of proteins with a molecular mass between 40 and 70 kDa were seen at the level of viral DNA peaks, some of which may represent catalytically active breakdown products of the TI enzyme. Large protein complexes were observed under the electron microscope in association with the viral chromosomes and appear to correspond to the SV40 DNA replication complex, including TI. Our results suggest that TI activity is indeed associated with the viral minichromosomes undergoing replication in vivo.
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PMID:DNA topoisomerase activity associated with simian virus 40 nucleoprotein complexes. 784 Sep 39

Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II-containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II-containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II-containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).
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PMID:An RNase-sensitive particle containing Drosophila melanogaster DNA topoisomerase II. 808 68

Three aspects of DNA topology were examined in two human squamous cell carcinoma lines of differing radiosensitivity (SQ-9G, D0 = 1.46 Gy; and SQ-20B, D0 = 2.36 Gy). High-salt-extracted nuclei (nucleoids) were taken from gamma-irradiated cells, stained with ethidium bromide and examined by flow cytometry. After 5 Gy, nucleoids from SQ-9G cells became 30% less efficient at adopting positive DNA supercoils than were unirradiated controls. In contrast, only a 4% difference was found with the radioresistant SQ-20B line. Both lines produced positive supercoils more efficiently after irradiation if first exposed to the topoisomerase II inhibitor VP16. Ethidium bromide titration of nucleoids was consistent with each containing similar numbers and sizes of DNA loops. In each line approximately 30-35% of DNA was accessible to trioxsalen, as shown by inter-strand crosslinking after UV photo-activation. Exhaustive digestion of nuclear DNA by DNase I removed more DNA from the radiosensitive than from the radioresistant cell line (12% vs 28% remaining). This difference was thought to be due to the increased accessibility of SQ-9G DNA in vitro. We suggest that a looser association of SQ-9G DNA with the nuclear matrix both promotes DNase I digestion and affects the ability of SQ-9G nucleoids to maintain positive DNA supercoils after irradiation. These data implicate the DNA matrix attachment region in the expression of radiation sensitivity in the cell lines studied.
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PMID:A correlation between DNA-nuclear matrix binding and relative radiosensitivity in two human squamous cell carcinoma cell lines. 809 63

We examined the promoter of the human type-I-DNA topoisomerase gene (hTOP1) for regions protected against DNase I digestion by nuclear proteins from HeLa or from adenovirus-transformed 293 cells. We identified ten protected DNA sequences within 580 bp of DNA upstream of the transcriptional-start sites and one additional site, which is located between the two clusters of transcriptional-start sites. Several of these protein-binding sites have significant similarities to recognition sequences of known transcription factors including factors Sp1, octamer transcription factor, cAMP-responsive-element-binding protein (CREB/ATF), NF-kappa B and members of the Myc-related family of basic/helix-loop-helix/leucine-zipper proteins. Other protein-binding sites show less or no similarities to known consensus sequences. We investigated the physiological significance of these protein-binding sites using a set of deletion and nucleotide-exchange mutants. We conclude that the expression of the hTOP1 gene is regulated by a complex network of negatively and positively acting transcription factors.
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PMID:The promoter region of the human type-I-DNA-topoisomerase gene. Protein-binding sites and sequences involved in transcriptional regulation. 822 37

We have investigated the effect of mAMSA, a potent topoisomerase II inhibitor, on the c-myc proto-oncogene of the acute promyelocytic leukemia HL60 cell line during its differentiation. When HL60 cells were induced by dimethylsulfoxide (DMSO) to terminally differentiate, a rapid drop in the level of c-myc mRNA was observed, followed by an arrest of cell proliferation. In contrast, the level of topoisomerase II mRNA was transiently increased with a maximum at 6 h after DMSO addition and was then completely abolished after 48 h, indicating that topoisomerase II is activated during the onset of HL60 differentiation. In exponentially growing cells, treatment by mAMSA results in the formation of topoisomerase II-mediated double strand DNA breaks in the c-myc gene at positions where topoisomerase II would normally nick and reseal the two strands. In HL60 cells treated with both mAMSA and DMSO, the sites in the c-myc gene at which mAMSA had induced cleavage were not altered. However, a DNA cleavage site located at the end of the first c-myc exon (position +3100), was strongly stimulated by mAMSA and DMSO treatment. This site fell within a DNase I hypersensitive region encompassing the MYC intron factor 1 (MIF1) binding site, where transcription elongation is normally blocked during differentiation. These data indicate that a change of topoisomerase II binding to critical regulatory region of the c-myc gene is associated with the downregulation of this gene during differentiation.
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PMID:Analysis of topoisomerase II-mediated DNA cleavage of the c-myc gene during HL60 differentiation. 824 49

Chromatin of rat elongating spermatids, steps 12-13, is distinguished by the replacement of histones with transition proteins and the presence of nicks within its DNA which are formed by an endogenous nuclease, possibly DNA topoisomerase II (topo II). Using an affinity-purified anti-topo II antibody, protein bands of approximately 161 and approximately 137 kDa were detected on immunoblots of pachytene spermatocytes and elongating spermatids, respectively. In cryosections, topo II was localized to meiotic chromosomes of pachytene spermatocytes and to nuclei of elongating spermatids. Extracts of isolated testicular nuclei and sonication-resistant spermatid nuclei (steps 12-19) demonstrated topo II activity as determined by the decatenation of kinetoplast DNA. The potential relationship between nucleoprotein changes during spermatogenesis and the formation of nicks was also examined. Heterogeneous testicular and sonication-resistant spermatid nuclei were treated with 0.8 mM protamine, followed by nick translation in the absence of DNase I. In both cases, there was a dramatic decrease in DNA polymerase I-dependent label incorporation. To determine whether or not endogenous nicks were present in mature sperm, but were inaccessible due to protamine-DNA interactions, epididymal sperm were extracted with high salt-dithiothreitol, followed by nick translation in the absence or presence of DNase I. Extracted sperm nuclei did not nick translate in the absence of DNase I; however, incorporation increased with increasing concentrations of DNase I, indicating that endogenous nicks were repaired prior to the completion of spermatogenesis. These and previously published results suggest that topo II in elongating spermatids may be involved in the DNA alterations that take place during spermatogenesis, including changes in DNA topography, repair, and loop formation, and may serve as a component of the nuclear matrix. The temporal appearance and disappearance of endogenous nicks may reflect the changes that elongating spermatid DNA undergoes as a consequence of alterations in nucleoprotein composition to establish the condensed state of the mature spermatozoon.
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PMID:Nicking of rat spermatid and spermatozoa DNA: possible involvement of DNA topoisomerase II. 839 68

The members of the human beta globin gene family are flanked by strong DNase I hypersensitive sites. The collection of sites 5' to the epsilon globin gene is able to confer high levels of expression of linked globin genes, but a function has not been assigned to the site 3' to the beta globin gene (3'HS1). Our analysis of this DNase I super hypersensitive site shows that the region is composed of multiple DNase I sites. By examination of the DNA sequence, we have determined that the region is very A/T-rich and contains topoisomerase II recognition sequences, as well as several consensus binding motifs for GATA-1 and AP-1/NF-E2. Gel mobility shift assays indicate that the region can interact in vitro with GATA-1 and AP-1/NF-E2, and functional studies show that the region serves as a scaffold attachment region in both erythroid and nonerythroid cell lines. Whereas many of the physical features of 3'HS1 are shared by 5'HS2 (a component of the 5' locus control region), transient expression studies show that 3' HS1 does not share the erythroid-specific enhancer activity exhibited by 5'HS2.
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PMID:Characterization of the DNase I hypersensitive site 3' of the human beta globin gene domain. 849 Jan 85

Polynuceosomes are constrained into loops or domains and are insulated from the effects of chromatin structure and torsional strain from flanking domains by the cross-complexation of matrix-attached regions (MARs) and matrix proteins. MARs or SARs have an average size of 500 bp, are spaced about every 30 kb, and are control elements maintaining independent realms of gene activity. A fraction of MARs may cohabit with core origin replication (ORIs) and another fraction might cohabit with transcriptional enhancers. DNA replication, transcription, repair, splicing, and recombination seem to take place on the nuclear matrix. Classical AT-rich MARs have been proposed to anchor the core enhancers and core origins complexed with low abundancy transcription factors to the nuclear matrix via the cooperative binding to MARs of abundant classical matrix proteins (topoisomerase II, histone H1, lamins, SP120, ARBP, SATB1); this creates a unique nuclear microenvironment rich in regulatory proteins able to sustain transcription, replication, repair, and recombination. Theoretical searches and experimental data strongly support a model of activation of MARs and ORIs by transcription factors. A set of 21 characteristics are deduced or proposed for MAR/ORI sequences including their enrichment in inverted repeats, AT tracts, DNA unwinding elements, replication initiator protein sites, homooligonucleotide repeats (i.e., AAA, TTT, CCC), curved DNA, DNase I-hypersensitive sites, nucleosome-free stretches, polypurine stretches, and motifs with a potential for left-handed and triplex structures. We are establishing Banks of ORI and MAR sequences and have undertaken a large project of sequencing a large number of MARs in an effort to determine classes of DNA sequences in these regulatory elements and to understand their role at the origins of replication and transcriptional enhancers.
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PMID:Chromatin domains and prediction of MAR sequences. 857 83

We have analyzed the topoisomerase II cleavage sites in the extrachromosomal ribosomal DNA of the lower eukaryote Physarum polycephalum using the topoisomerase II-specific inhibitor, 6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid. Most of the in vivo topoisomerase II cleavage sites were found either in the transcribed region of ribosomal DNA or in the palindromic region surrounded by the replication origins. Two classes of sites were identified: those which correlate with DNase I hypersensitive sites and corresponding to an open chromatin configuration (transcribed region) and internucleosomal cleavage sites (in the region of replication origins). Topoisomerase II drug-induced cleavage in the ribosomal DNA was considerably reduced upon Physarum differentiation to a dormant stage of life, the spherules. In contrast, the amount of drug-dependent cleavage was found to increase during the metaphase of mitosis, when rDNA transcription is shut off. These findings suggest a role for topoisomerase II in the ribosomal DNA minichromosomes segregation, in addition to its role in transcription. Finally, the similarity between in vivo sites and those observed following drug treatment of isolated nuclei indicates that no profound change occurs in rDNA chromatin conformation during nuclei isolation. By contrast, in vitro cleavage sites with purified topoisomerase II weakly correlate to in vivo, indicating a prominent role for chromatin structure in determining the interaction sites of topoisomerase II with DNA in vivo.
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PMID:In vivo topoisomerase II cleavage sites in the ribosomal DNA of Physarum polycephalum. 863 39

Most developing lymphocytes spontaneously die in the thymus during positive and negative selection of the T cell repertoire. By evaluating the expression of the proliferation antigens Ki-67 and PCNA, we demonstrated here that more than 95% of thymocytes are potentially proliferating. The coincidence within the same cell population of death and proliferation is thus apparent in developing thymocytes. Using dual-parameter cytometric techniques to evaluate in single cells the amount of DNA versus light-scattering values, we found that spontaneous thymocyte apoptosis occurs with similar frequency in all the cycle phases, whereas apoptosis induced by the anti-topoisomerase-II, etoposide (which is the consequence of irreversible DNA damage), takes place with higher frequency in S and G2 phases (i.e., in those cycle phases in which DNA is subjected to torsional constraints). The capability of thymocytes to enter apoptosis was also monitored by digesting DNA in situ with DNase I (a nuclease that cleaves DNA mimicking the nuclear damage common to most apoptotic suicides). We also show that endonuclease-mediated DNA digestion occurs to a similar extent in cells with different DNA contents, i.e., in cycle phases in which the superstructural organization of chromatin is markedly different.
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PMID:Spontaneous apoptosis of thymocytes is uncoupled with progression through the cell cycle. 898 20


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