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)

To study a possible differential involvement of type I and type II DNA topoisomerases in the functional and structural changes that chromatin undergoes during spermatogenesis, we have determined both enzymatic activities in chicken testis cell nuclei at successive stages of differentiation. Whereas DNA topoisomerase I varies in parallel with transcriptional activity, DNA topoisomerase II was present in both replicating, transcriptionally active chicken testis cells and nonreplicating, transcriptionally inactive late spermatids. The presence of DNA topoisomerase II activity in late spermatids and, in addition, the relative increment of drug-induced topo-II-mediated DNA cleavage detected in these cells, suggest that DNA topoisomerase II might modulate the topology of DNA during the marked changes that chromatin structure undergoes in the nucleohistone-nucleoprotamine transition at the end of the spermiogenesis and could be involved in the final organization of DNA within the nucleus of the male gamete.
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PMID:DNA topoisomerase II activity in nonreplicating, transcriptionally inactive, chicken late spermatids. 254 58

We have examined the roles of eukaryotic DNA topoisomerases I and II in DNA replication by the use of a set of four isogenic strains of Saccharomyces cerevisiae that are TOP1+ TOP2+, TOP1+ top2 ts, delta top1 TOP2+, and delta top1 top2 ts. Cells synchronized by treatment with the alpha-mating factor, or by cycles of feeding and starvation, were released from cell-cycle arrest, and the size distribution of DNA chains that were synthesized after the cells reentered the S-phase was determined as a function of time. The results indicate that synthesis of short DNA chains several thousand nucleotides in length can initiate in the absence of both topoisomerases, but their further elongation requires at least one of the two topoisomerases. Inactivation of DNA topoisomerase II does not alter significantly the time dependence of the patterns of nascent DNA chain synthesis, which is consistent with the notion that the requirement of this enzyme for viability is due to its essential role during mitosis, when pairs of intertwined newly replicated chromosomes are being segregated. The absence of DNA topoisomerase I leads to a temporary delay in the extension of the short DNA chains; this delay in chain elongation is also reflected in the rate of total DNA synthesis in the delta top1 mutant during the early S-phase. Thus, in wild-type cells, DNA topoisomerase I is probably the major replication swivel. The patterns of DNA synthesis in asynchronously grown delta top1 top2 ts cells at permissive and non-permissive temperatures are also consistent with the above conclusions.
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PMID:Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae. 254 54

Limited digestion of E. coli DNA topoisomerase I with trypsin or papain generated a DNA-binding domain of MW 14,000 corresponding to the carboxyl terminal of the enzyme. This fragment binds to single-stranded DNA agarose as tightly as the intact enzyme. It required around 400 mM NaCl for elution. A truncated topoisomerase that lacks this C-terminal domain was purified. It was eluted from the single-stranded DNA agarose column at around 150 mM NaCl. Although the truncated enzyme could relax negatively supercoiled DNA as efficiently as the intact enzyme at low ionic strength, its processivity was more sensitive to increasing salt concentration. Measurement of binding to fluorescent etheno-M13 DNA also demonstrated that the presence of the C-terminal domain confers higher affinity to DNA for the enzyme.
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PMID:The carboxyl terminal domain of Escherichia coli DNA topoisomerase I confers higher affinity to DNA. 256 Jan 91

Chronic lymphocytic leukemia is a neoplastic disease in which drug resistance invariably occurs. We have studied the uptake and interaction with molecular targets of two drugs, chlorambucil and adriamycin, in CLL lymphocytes and CHO cell lines. Resistance does not appear related to uptake for either drug. Exposure to CLB causes DNA cross-links in the sensitive but not in the resistant cell line. The GSH content of B-CLL lymphocytes is depleted after a 20-hr incubation. An inability to maintain its GSH content may contribute to this cell's vulnerability to CLB. The resistance of CLL lymphocytes to ADR may be related to the undetectable levels of its target enzyme DNA topoisomerase II. Future approaches may involve study of novel anthracyclines, DNA topoisomerase I inhibitors and the development of in vitro predictive tests.
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PMID:Studies on drug resistance in chronic lymphocytic leukemia. 256 Dec 48

K6-1 and 50B-3 cell lines, resistant to VP-16, a DNA topoisomerase II inhibitor, were established from two different types of cells respectively: human T-cell derived acute lymphoblastic leukemia cell line RPMI8402 and mouse mammary tumor cell line FM3A. IC50 values of K6-1 and 50B-3 cells to VP-16, evaluated by the colony forming ability on methyl cellulose medium, were 11- and 84-fold higher than their sensitive parental cell lines, respectively. Membrane permeability of the drug was not responsible for the resistance in K6-1 and 50B-3 cells. Quantitative analysis of drug-induced DNA cleavage (so called cleavable complex formation) was performed using 32P end-labeled pBR322 restriction fragments. The formation of the topoisomerase II-DNA cleavable complex stimulated by VP-16 in 50B-3 cells was approximately 1/5 compared with that of FM3A wild-type cells. Dot blot analysis of RNA extracted from these cell lines showed that the levels of mRNA for DNA topoisomerase II in 50B-3 cells were markedly decreased and that catalytic activity was reduced to 1/2-1/3 compared with that of parent cells. There was a slight reduction of DNA topoisomerase II mRNA in K6-1 cells. However, DNA topoisomerase II activities were similar in wild-type and K6-1 cells. In addition, 50B-3 cells showed cross resistance to VM-26, m-AMSA and adriamycin, whereas K6-1 cells exhibited increased resistance only to VM-26. These resistant cell lines did not show collateral sensitivity to CPT-11, a DNA topoisomerase I inhibitor. Southern blot analysis of genomic DNA did not show any change in the restriction pattern of the DNA topoisomerase II gene between the parental and their resistant lines. These findings suggest that the reduced levels in DNA topoisomerase II contribute to the drug resistance of 50B-3 cells.
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PMID:DNA topoisomerase: the mechanism of resistance to DNA topoisomerase II inhibitor VP-16. 256 62

Camptothecin is a cytotoxic drug which inhibits cellular nucleic acid synthesis. Associated with this inhibition is the induction of protein-linked DNA strand breaks. Recent studies have demonstrated that camptothecin interferes with the DNA breakage and rejoining activity of the enzyme DNA topoisomerase I and stabilizes a cleavable complex between this enzyme and DNA. Treatment of this complex with a protein denaturant results in DNA strand breaks and the covalent attachment of topoisomerase to the 3'-end of the DNA breaks. In this paper we have mapped the camptothecin-induced DNA breaks on the hsp 70 heat-shock gene in cultured Drosophila cells. Drug-induced breaks are present primarily within the coding region of this gene and occur only when transcription of this gene is activated by heat. Camptothecin (20 microM) was also observed to inhibit heat-induced hsp 70 transcription greater than 70%. The possible role of topoisomerase I in hsp 70 heat-shock gene transcription is discussed.
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PMID:Camptothecin inhibits hsp 70 heat-shock transcription and induces DNA strand breaks in hsp 70 genes in Drosophila. 281 31

Transcription of a right-handed double-helical DNA requires a relative rotation of the RNA polymerase and its nascent RNA around the DNA. We describe conditions under which the resistance to the rotational motion of the transcription ensemble around the DNA can be large. In such cases, the advancing polymerase generates positive supercoils in the DNA template ahead of it and negative supercoils behind it. Mutual annihilation of the positively and negatively supercoiled regions may be prevented by anchoring points on the DNA to a large structure, or, in the case of an unanchored plasmid, by the presence of two oppositely oriented transcription units. In prokaryotes, DNA topoisomerase I preferentially removes negative supercoils and DNA gyrase (topoisomerase II) removes positive ones. Our model thus provides an explanation for the experimentally observed high degree of negative or positive supercoiling of intracellular pBR322 DNA when DNA topoisomerase I or gyrase is respectively inhibited. We discuss the implications of our model in terms of supercoiling regulation, DNA conformational transitions, and gene regulation in both prokaryotes and eukaryotes.
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PMID:Supercoiling of the DNA template during transcription. 282 50

DNA topoisomerase mutants of Escherichia coli and Saccharomyces cerevisiae were used to study the topological state of intracellular DNA. In E. coli, it is shown that switching off the gene topA encoding DNA topoisomerase I leads to an increase in the degree of negative supercoiling of intracellular DNA and inhibition of the growth of the cells: a d(pCpG)16.d(pCpG)16 sequence on a plasmid is also shown to flip from a right-handed B-helical structure to a left-handed Z-helical structure in vivo when topA is switched off. In S. cerevisiae, the topological state of intracellular DNA is little affected by the cellular levels of the topoisomerases.
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PMID:DNA supercoiling in vivo. 283 49

DNA topoisomerases I and II are nuclear enzymes which modify DNA topology by their ability to break and reseal one or both strands in concert. Each of these enzymes has important functions in DNA replication, and very likely in other genetic processes as well. In addition, each can serve as a cancer chemotherapy target. The plant alkaloid camptothecin traps DNA topoisomerase I in a form which is covalently linked to DNA. Presumably this action is the basis for its anti-tumor effect, although this has not been conclusively demonstrated. The evidence for DNA topoisomerase II as a target for intercalating agents and epipodophyllotoxins is more formidable. Each of these classes of agents traps the enzyme on DNA in a structure referred to as a 'cleavable complex'. Illicit recombination events, as well as induction of an 'SOS-like' response analogous to that found in bacteria, have been suggested as possible mechanisms for cell death following cleavable complex formation. Development of new agents directed at topoisomerase II will depend heavily on understanding the nature of the interaction between the drug, enzyme and DNA. Hypotheses are presented in this paper on this interaction. Intracellular content of topoisomerase II is linked to the ability of the cell to enter a G0-like state, and the inability of malignant cells to undergo such a change may provide part of the basis for therapeutic selectivity. The ability to modulate topoisomerase II levels may provide an opportunity for therapeutic intervention.
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PMID:DNA topoisomerases in cancer therapy. 283 59

The intracellular level of DNA topoisomerase II appears to be reversibly regulated by serum concentration in cultured primary human skin fibroblasts (HSF). Upon serum starvation, the intracellular level of topoisomerase II in HSF, as monitored by immunoblotting with antitopoisomerase II antibodies, gradually decreased to a nondetectable level (less than 10(4) copies/cell) over a period of 72 h. Addition of 10% serum to the starved cells led to a gradual increase of the intracellular topoisomerase II to the original level (approximately 10(6) copies/cell) over a period of 24 h. The intracellular DNA topoisomerase II level in HSF is also sensitive to cell density; minimally a 7-fold decrease was observed when HSF were grown to saturation density in a constant serum concentration. Similarly, the intracellular levels of DNA topoisomerase II in other "nontransformed" cells such as mouse NIH 3T3 and 3T6 cells are also sensitive to both the serum concentration and the cell density. In contrast, topoisomerase II levels in transformed cells such as HeLa cells, L1210 cells, and SV40 T-antigen-transformed COS-1 cells are maintained at high levels (approximately 10(6) copies/cell) and are much less sensitive to growth conditions. The topoisomerase II level in HeLa cells synchronized by a double thymidine block remained relatively constant (less than 2-fold difference) throughout the late G1, S, G2, and M phases of the cell cycle. Our results suggest that the level of DNA topoisomerase II is primarily regulated in the G0-G1 phase of the cell cycle and is elevated to a high level (approximately 10(6) copies/cell) in proliferating cells. In contrast, the intracellular levels of DNA topoisomerase I in these cells were largely unaffected by these growth conditions either in HSF or in HeLa cells.
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PMID:Proliferation-dependent regulation of DNA topoisomerase II in cultured human cells. 283 57


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