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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)

Since DNA topoisomerase II (EC 5.99.1.3) is an essential enzyme in yeast, heterologous topoisomerase II gene expression in yeast cells can provide a system for analyzing the structure and function of topoisomerase II genes from other species. A series of yeast expression plasmids was constructed in which segments of the cDNA sequences encoding Drosophila DNA topoisomerase II were inserted under the transcriptional control of yeast GAL1 promoter. Expression of the functional form of Drosophila topoisomerase II cDNA can complement conditionally lethal, temperature-sensitive mutations in the yeast topoisomerase II gene (TOP2), as well as mutations in which the TOP2 locus was disrupted. The survival of these yeast cells depends upon the continuous expression of Drosophila topoisomerase II. Repression of Drosophila gene expression by glucose causes these yeast cells to cease dividing after a few generations. In addition to these genetic complementation data, the expression of the Drosophila topoisomerase II gene in yeast cells with a disruption in TOP2 can also be detected by immunochemical methods with an antibody specific for Drosophila topoisomerase II.
Proc Natl Acad Sci U S A 1988 Sep
PMID:Functional expression of a Drosophila gene in yeast: genetic complementation of DNA topoisomerase II. 284 62

A topoisomerase has been purified from extracts of a topoisomerase I-deficient strain of Escherichia coli based solely on its ability to segregate pBR322 DNA replication intermediates in vitro. This enzyme rapidly decatenated multiply linked form II:form II DNA dimers to form II DNA, provided that the DNA substrate contained single-stranded regions. Efficient relaxation of negatively supercoiled DNA was observed when reaction mixtures were incubated at 52 degrees C, but not at 30 degrees C (the temperature at which decatenation was readily observed). This topoisomerase was insensitive to the DNA gyrase inhibitor norfloxacin and unaffected by antibody directed against topoisomerase I. Relaxation of a unique plasmid topoisomer revealed that this decatenase changed the linking number of the DNA in steps of one and was therefore a type 1 topoisomerase. The cleavage pattern of a fragment of single-stranded phi X174 DNA generated by this decatenase was virtually identical to that reported for topoisomerase III, the least characterized topoisomerase present in E. coli.
J Biol Chem 1988 Sep 15
PMID:Identification of a potent decatenating enzyme from Escherichia coli. 284 17

A novobiocin-resistant BHK cell line, designated as NovrA2, was found to exhibit cross-resistance to other topoisomerase II inhibitors such as 4'-dimethylepipodophyllotoxin-4-(4,6-O-ethylidine-beta-D-glu copyranoside) (VP-16), adriamycin, and 4'-(9-acridinyl-amino)methanesulfon-m-anisidide (m-AMSA), and also to different types of drugs such as vinblastine and arabinocytidine. Nalidixic acid-resistant cells (A2Nalr) of the NovrA2 cell line were phenotypically reverted to novobiocin sensitivity like wild-type cells and were also partially reverted to sensitivity to VP-16 and adriamycin, but not to vinblastine and arabinocytidine. When VP-16 was added to cell culture, the drug-induced DNA strand breaks were much fewer in NovrA2 cells than in BHK cells. This reduced level of strand breaks in NovrA2 cells was not due to reduced drug uptake, because the two cell lines accumulated similar levels of radiolabeled VP-16. VP-16 also induced fewer DNA breaks in isolated nuclei of NovrA2 cells than in those of BHK cells. There was no significant difference in the VP-16-induced DNA cleavage activities of partially purified topoisomerase II from BHK and Novr cells. These results show that the resistance of NovrA2 cells to various drugs is not acquired by a defense mechanism related to membrane permeability and suggest that the resistance of the NovrA2 cells to topoisomerase II inhibitors might be due in part to alteration in a topoisomerase II associated factor(s).
Somat Cell Mol Genet 1988 Sep
PMID:Cross-resistance of novobiocin-resistant BHK cell line to topoisomerase II inhibitors. 284 88

Endogenous topoisomerase II cleavage sites were mapped in the chicken beta A-globin gene of 12- to 14-day embryonic erythrocytes. A major topoisomerase II catalytic site was mapped to the 5' end of the globin gene which contained a nucleosome-free and DNase I-hypersensitive site and additional but minor sites were mapped to the second intron and 3' of the gene to a tissue-specific enhancer. Cleavage sites, mapped in situ by indirect end labeling, were aligned to single-base-pair resolution by comparison to a consensus sequence derived for vertebrate topoisomerase II catalytic sites. In contrast to embryonic erythrocytes, endogenous topoisomerase II cleavages were not detected in erythrocytes from peripheral blood of adult chickens; therefore, as the transcriptional activity of the beta A-globin gene declines during terminal differentiation of erythrocytes, the activity of topoisomerase II in situ declines as well, despite the fact that DNase I hypersensitivity persists. The results showed that DNase I-hypersensitive chromatin can be maintained in the absence of topoisomerase II activity and suggested that topoisomerase II acts at hypersensitive sites because of an inherent attraction to some preexisting combination of DNA sequence or chromatin structure associated with DNase I-hypersensitive regions.
Mol Cell Biol 1988 Sep
PMID:DNase I hypersensitivity is independent of endogenous topoisomerase II activity during chicken erythrocyte differentiation. 285 23

A cloned plasmid, pmyc(H-K), containing sequences derived from human c-myc gene replicated in vitro in Raji nuclear extract in a semiconservative manner. Using this system, it was found that phosphatidylinositol and cardiolipin strongly inhibited the replication of pmyc(H-K) in vitro, whereas other phospholipids, i.e., phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and sphingomyelin, had no appreciable effect. The concentrations of phosphatidylinositol and cardiolipin producing 50% inhibition of the replication were 4.6 and 5.4 microM, respectively. Phosphatidylinositol and cardiolipin inhibited the relaxation of pmyc(H-K) supercoiled DNA, but showed little or weaker effects on DNA polymerase alpha and topoisomerase II in Raji nuclear extract. These results suggest that phosphatidylinositol and cardiolipin antagonize the replication of pmyc(H-K) in vitro, through, at least in part, the interaction with topoisomerase I.
J Biochem 1988 Sep
PMID:Phospholipid modulates in vitro replication of autonomous replicating sequence from human cells. 285 2

Novobiocin inhibits DNA topoisomerases. It also inhibits excision repair of DNA photodamage, blocking both repair synthesis and the earlier step of incision at u.v. damage sites (as measured by the accumulation of DNA strand breaks in u.v.-irradiated interphase cells treated with DNA synthesis inhibitors such as hydroxyurea or cytosine arabinoside). It has been supposed, therefore, that novobiocin affects repair by blocking a putative topoisomerase step prior to incision. But we find that novobiocin also has a marked dose- and time-dependent effect on mitochondria: in cells exposed to novobiocin, mitochondria swell and their cristae become disrupted, and the intracellular ATP:ADP ratio is lowered, though the membrane potential is maintained as judged by rhodamine 123 fluorescence. Mitotic cells are more resistant to mitochondrial disruption by novobiocin than are interphase cells. This correlates with a relative resistance of u.v.-irradiated mitotic cells to the inhibition of incision by novobiocin. The chromosomal decondensation that results from the accumulation of DNA breaks due to incision when u.v.-irradiated mitotic cells are treated with hydroxyurea and cytosine arabinoside is largely suppressed by novobiocin. Furthermore, the suppression of induced strand break accumulation is partly due to a suppression by novobiocin of the uptake and phosphorylation of cytosine arabinoside; breaks accumulated in u.v.-irradiated cells in the presence of aphidicolin, an inhibitor of DNA polymerase alpha that does not require phosphorylation, are less novobiocin-sensitive. We conclude that the effects of novobiocin on excision repair are more likely to be due to a non-specific effect on ATP metabolism than to a specific effect on a repair-related topoisomerase.
Carcinogenesis 1985 Sep
PMID:Novobiocin inhibition of DNA excision repair may occur through effects on mitochondrial structure and ATP metabolism, not on repair topoisomerases. 299 34

Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon.
J Mol Biol 1985 Sep 05
PMID:DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling. 299 87

A Rhodopseudomonas capsulata nifH::lacZ gene fusion was used to isolate constitutive mutants of R. capsulata, unable to repress nif gene transcription anaerobically with every fixed-nitrogen source tested. When these nifc strains were grown aerobically, nif gene transcription was repressed. These results indicate that the regulation of nif gene transcription by fixed nitrogen is different from the regulation by oxygen. Under anaerobic conditions, nif gene transcription in both R. capsulata and Klebsiella pneumoniae is specifically prevented by inhibitors of DNA gyrase [DNA topoisomerase type II (ATP-hydrolyzing), EC 5.99.1.3]. A recent study has shown that anaerobically grown Salmonella typhimurium have high DNA gyrase activity, whereas aerobically grown cells have high DNA topoisomerase type I (EC 5.99.1.2) activity and DNA that is more relaxed [Yamamoto, N. & Droffner, M. L. (1985) Proc. Natl. Acad. Sci. USA 82, 2077-2081]. In view of these results, we suggest that the control of nif gene transcription in response to oxygen is determined by the action of DNA gyrase and DNA topoisomerase I. Thus, although nitrogen control of nif gene expression requires the products of regulatory genes for which constitutive mutations can be isolated, oxygen appears instead to prevent the adoption of a DNA conformation necessary, directly or indirectly, for nif gene transcription.
Proc Natl Acad Sci U S A 1986 Sep
PMID:Anaerobic regulation of nitrogen-fixation genes in Rhodopseudomonas capsulata. 301 47

T4 gene 52 encodes one of the three subunits of T4 DNA topoisomerase. The T4 enzyme is required for normal phage DNA replication. I have cloned the entire gene, and it is expressed in uninfected E. coli cells. The sequence of 1966 nucleotides of T4 deletion delta sa9 surrounding gene 52 has been determined. The reading frame of the gene was established by identifying the first ten amino acids in the large open reading frame derived from the DNA sequence as those at the amino-terminus of the purified 52-protein. Based on the DNA sequence, 52-protein has 441 amino acids and a calculated peptide molecular weight of 50,583 daltons. This T4 topoisomerase subunit shares significant amino acid sequence homology with the gyrA subunit of bacterial gyrases and the carboxyl-half of yeast topoisomerase II in spite of the large differences in their sizes, confirming their functional equivalence in type II enzyme directed DNA topoisomerization. Amino acid sequence homology is highest in the amino-terminal portions of the equivalent peptides. The homology alignment suggests a consensus sequence organization surrounding the reactive tyrosine which is used to form the transient protein-DNA intermediate in the double-stranded DNA passing reaction. The delta sa9 deletion in T4 brings gene 52 to a location 30 nucleotides 3' from the rIIB gene whose C-terminal 167 codons are also reported here.
Nucleic Acids Res 1986 Sep 25
PMID:The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase. 302 May 13

We have analyzed 1 kb of cloned human c-fos sequence (-711 to +287) for calf thymus DNA topoisomerase II cleavage sites in vitro. Using the anti-tumor drug VP16 (demethylepipodophyllotoxin-beta-D-glucoside) with purified topoisomerase II, we identify twelve sites. Five sites are clustered around position -306 in a region that possesses enhancer-like properties. A second cluster of three sites is positioned 15 bp upstream of the TATA promoter element. With a HeLa nuclear extract as a source of topoisomerase II, a subset of cleavage sites is conserved within the two clusters. The cleavage sites in the enhancer-like element are conserved in the homologous region of the murine c-fos. These findings raise the possibility that topoisomerase II is involved in mediation of mitogen-induced c-fos expression.
EMBO J 1986 Sep
PMID:DNA topoisomerase II cleaves at specific sites in the 5' flanking region of c-fos proto-oncogenes in vitro. 302 64


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