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)

Extracts of Drosophila embryos can mediate the assembly of a chromatinlike structure from histones and DNA under physiological conditions. The histone-DNA complex formed in vitro contains micrococcal nuclease-sensitive sites spaced at 200-base pair intervals. More extensive digestion of the complex by micrococcal nuclease generates 11S particles which cosediment with nucleosome core particles isolated from native chromatin. These particles contain 140-base pair DNA fragments which upon further cleavage with micrococcal nuclease give rise to a pattern of discretely sized DNA fragments characteristic of nucleosome core particles. We have assayed the chromatin assembly process both qualitatively by measuring the induction of supertwists into a relaxed circular DNA (a process requiring a nicking-closing enzyme) and quantitatively by measuring the formation of micrococcal nuclease-resistant DNA fragments from radioactively labeled linear DNA. The amount of chromatin formed depends primarily on the amount of histones, whereas the rate of assembly depends on the amount of extract protein added. The factors in the extract that mediate chromatin assembly appear to interact first with the DNA because preincubation of the DNA with the extract markedly increases the extent of assembly.
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PMID:Extracts of Drosophila embryos mediate chromatin assembly in vitro. 11 49

During transcription, positive DNA supercoils generated ahead of RNA polymerase could theoretically uncoil the negative DNA supercoils associated with nucleosomes and thereby decondense the chromatin fiber in preparation for RNA polymerase passage. Here we examine the effect of positive DNA supercoiling on the structure of yeast 2-microns minichromosomes. We utilized a conditional topoisomerase mutant expressing Escherichia coli topoisomerase I to convert the DNA supercoiling state from negative to positive in vivo. Minichromosomes containing positively supercoiled DNA exhibited a striking increase in DNase I sensitivity. They also displayed additional micrococcal nuclease cleavage sites but yielded nearly typical nucleosomal ladders after extensive digestion. Upon in vitro relaxation with eukaryotic topoisomerase I, the minichromosomes remained DNase I sensitive but were converted to negative DNA supercoiling with a slightly increased linking number compared to typical minichromosomes, thus indicating the presence of bound histones. Therefore, positive DNA supercoiling provides a mechanism for generating, but is not required for maintaining, a conformation in chromatin characteristic of highly transcribed genes.
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PMID:Positive DNA supercoiling generates a chromatin conformation characteristic of highly active genes. 194 86

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.
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PMID:Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin. 216 70

Specific inhibitors of eukaryotic DNA topoisomerases I and II (camptothecin and VM-26, respectively) were used to examine the involvement of topoisomerases in DNA replication and chromatin assembly in vivo. When used singly, either camptothecin or VM-26 inhibited DNA synthesis in HeLa cells by more than 80%; when used simultaneously, the inhibitors effectively stopped replication, demonstrating that at least one class of topoisomerase must be active for fork propagation in vivo. To study nucleosome assembly during topoisomerase inhibition, three experimental strategies were employed: (1) pulse-chase experiments; (2) analyses of chromatin synthesized during residual replication in the presence of either camptothecin or VM-26; and (3) the assembly of previously replicated, unassembled DNA, generated in the presence of protein synthesis inhibitors. Using sensitivity to micrococcal nuclease and the maturation of non-nucleosomal replication intermediates as criteria, neither camptothecin nor VM-26, alone or in concert, inhibited nucleosome assembly under any experimental protocol tested. These data provide evidence that, although topoisomerase activity is essential for DNA replication, neither continuous fork propagation nor topoisomerase activity is required for chromatin assembly on new DNA.
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PMID:Inhibitors of topoisomerases I and II arrest DNA replication, but do not prevent nucleosome assembly in vivo. 255 22

Changes in the association of the catalytic subunit and the regulatory subunits of isozymes I and II of cAMP-dependent protein kinases (RI and RII, respectively) with the transcriptionally active chromatin fraction from rat liver were examined after a glucagon/theophylline injection and also after partial hepatectomy. Chromatin was partitioned into transcriptionally active and bulk, transcriptionally inactive fractions by digestion with micrococcal nuclease under appropriate conditions. In both experimental models, an increased content of catalytic and both RI and RII subunits was observed in chromatin fractions that were enriched in transcriptionally active DNA, particularly in the fraction associated with the residual nuclear matrix-lamina. The changes in the association of the subunits with these fractions paralleled the increases in intracellular cAMP levels and occurred in a time frame compatible with the changes in gene expression. The catalytic subunits could be removed from the nuclear matrix-lamina fraction by salt, whereas the two regulatory subunits remained tightly bound. The data support the concept of a direct role of the regulatory subunits of cAMP-dependent protein kinases in the induction of gene expression. However, we were unable to confirm that RII possessed an intrinsic topoisomerase activity.
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PMID:The regulatory and catalytic subunits of cAMP-dependent protein kinases are associated with transcriptionally active chromatin during changes in gene expression. 289 95

We have examined the long-range distribution of double-stranded DNA cleavage sites for Drosophila melanogaster topoisomerase II. These studies reveal a novel partitioning of preferred topoisomerase II cleavage sites. In the eukaryotic DNAs examined, major cleavage sites were typically found in nontranscribed spacer segments and close to the 5' and 3' boundaries of genes. In contrast, there were few if any prominent cleavage sites within genes. In addition, most of the major topoisomerase II cleavage sites closely corresponded to naked DNA hypersensitive sites for the prokaryotic enzyme, micrococcal nuclease.
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PMID:Novel partitioning of DNA cleavage sites for Drosophila topoisomerase II. 298 70

Newly-repaired DNA in chromatin is more sensitive to micrococcal nuclease than bulk DNA, but tends to become equally sensitive with time. This rearrangement of chromatin, which had previously been observed following repair of lesions produced by UV-light and of some bulky adducts, has now been shown to occur after repair of lesions induced by hydrogen peroxide and dimethylsulfate. In both cases there was an enhanced sensitivity to nuclease digestion of newly repaired DNA followed by a rearrangement whose kinetics was very similar to that observed in UV irradiated cells. Benzamide and 3-aminobenzamide, inhibitors of the synthesis of poly (ADP-ribose) and novobiocin, an inhibitor of topoisomerase, had no effect on the initially enhanced digestibility of repaired regions or on the chromatin rearrangement that followed. Poly (ADP-ribose) polymerase and topoisomerase are known to play some role in excision repair and have also been shown to cause alterations in the chromatin structure. However, the present results show that these alterations are not involved in this kind of chromatin rearrangement.
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PMID:Rearrangement of mammalian chromatin structure following excision repair: absence of an effect of inhibitors of poly (ADP-ribose) polymerase and topoisomerase. 301 3

Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.
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PMID:Simian virus 40 minichromosomes contain torsionally strained DNA molecules. 301 97

By autodigestion (endogenous endonucleolysis) of rat liver (RL) or rat-ascites hepatoma (AH) nuclei, the nucleosomes were released from the RL, but not from the AH, nuclei. In contrast, by micrococcal nuclease digestion (exogenous endonucleolysis), the nucleosomes were released more rapidly from the AH than from the RL nuclei. A 0.6 M NaCl extract of the RL or AH nuclei was filtered through a Sephadex G-100 column. The resulting topoisomerase fraction was subjected to DNA relaxation and catenation assays with pBR322 DNA as a substrate. Consequently, the relaxation activity was almost the same between the RL and AH fractions, whereas the catenation activity was much higher in the AH fraction.
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PMID:Endonucleolytic activities of nuclei from rat ascites hepatoma. 302 20

We have previously constructed a yeast strain (UKY403) whose sole histone H4 gene is under control of the GAL1 promoter. This yeast arrests in G2 upon glucose treatment as a result of histone H4 depletion. The yeast PHO5 gene contains phase nucleosomes covering promoter (UAS) sequences in the PHO5 repressed state and it has been suggested that nucleosomes prevent the binding of positively acting factors to these UAS sequences. Using UKY403 we examined the length of polynucleosomes and nucleosome phasing in the PHO5 upstream region by the use of micrococcal nuclease and indirect end-labeling. It was found that glucose arrest led to a severe disruption in PHO5 chromatin structure and that most nucleosomes had their position altered or were lost from the PHO5 promoter region. Cell undergoing nucleosome depletion synthesized large quantities of accurate PHO5 transcripts even under repressive, high inorganic phosphate conditions. Histone H4 depletion did not appear to affect the repression or activation of another inducible yeast gene, CUP1. Arrest with landmarks in early G1 (in the cell division cycle mutant cdc28) or in various stages of G2 (in cdc15, cdc17 and cdc20) does not activate PHO5; nor does arrest due to chromosome topology changes (in top2 or the top1top2 topoisomerase mutants). cdc14, which has its arrest landmark at a similar point in the cell cycle as cdc15, does derepress PHO5. However, since it also leads to derepression of CUP1 it is probably functioning through an independent mechanism. Therefore, our data suggest that nucleosomes regulate PHO5 transcription.
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PMID:Depletion of histone H4 and nucleosomes activates the PHO5 gene in Saccharomyces cerevisiae. 304 34


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