Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The binding of purified Drosophila topoisomerase II to the highly bent DNA segments from the SV40 terminus of replication and C. fasciculata kinetoplast minicircle DNA (kDNA) was examined using electron microscopy (EM). The probability of finding topoisomerase II positioned at or near the bent SV40 terminus and Crithidia fasciculata kDNA was two- and threefold higher, respectively, than along the unbent pBR325 DNA into which the elements had been cloned. Closer examination demonstrated that the enzyme bound preferentially to the junction between the bent and non-bent sequences. Using gel electrophoresis, a cluster of strong sodium dodecyl sulfate-induced topoisomerase II cleavage sites was mapped to the SV40 terminus DNA, and two weak cleavage sites to the C. fasciculata kDNA. As determined by EM, Drosophila topoisomerase II foreshortened the apparent length of DNA by only 15 base-pairs when bound, arguing that it does not wrap DNA around itself. When bound to pBR325 containing the C. fasciculata kDNA and the SV40 terminus, topoisomerase II often produced DNA loops. The size distribution was that predicted from the known probability of any two points along linear DNA colliding. In vitro mapping of topoisomerase II on DNA whose ends were blocked by avidin protein revealed that binding is enhanced at sites located near a blocked end as compared to a free end. These observations may contribute towards establishing a framework for understanding topoisomerase II-DNA interactions.
J Mol Biol 1991 Jan 05
PMID:Drosophila topoisomerase II-DNA interactions are affected by DNA structure. 184 28

The administration of the DNA topoisomerase II inhibitors 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) (10(-7) M), VP-16 (2 x 10(-7) M), or novobiocin (1.5 x 10(-4) M) reduces the growth activity of human promonocytic leukemia U-937 cells, by arresting them preferentially at the G2 (m-AMSA and VP-16) or at the G1 and G2 (novobiocin) phases of the cell cycle. Under these conditions, m-AMSA and VP-16 induce the differentiation of the cells efficiently, as proved both by an increase in the production of reactive oxygen species and by the activation of the surface expression of CD11b and CD11c, two differentiation-specific antigens. Novobiocin also induces the expression of those differentiation markers, but to a lesser extent. Analyses by Northern blot indicate that the topoisomerase II inhibitors reduce the levels of c-myc and beta-actin mRNA and increase the levels of vimentin mRNA. The expression of vimentin is also stimulated at the protein level, as indicated by immunofluorescence assays. This represents one of the few known instances in which topoisomerase inhibitors stimulate gene expression in eukaryotic cells.
Mol Pharmacol 1991 Apr
PMID:Differentiation of human promonocytic leukemia U-937 cells with DNA topoisomerase II inhibitors: induction of vimentin gene expression. 185 89

A high molecular weight mitochondrial DNA (mtDNA) replication complex, associated with the mitochondrial membrane, was isolated by sucrose gradient centrifugation from purified wheat embryo mitochondria. This complex comprised the mtDNA as well as enzyme activities involved in the replication and transcription of the organelle genome, such as DNA polymerase, RNA polymerase and topoisomerase type I. The isolated complex is active in mtDNA and mtRNA synthesis in vitro. Electron microscopy and lipid analysis confirmed the membrane origin of this complex. Enzyme activities are resistant to physiological ionic strengths, 0.1-0.2 M KC1, while the membrane-mtDNA association is resistant up to 1 M KC1. DNase treatment of the complex released the DNA polymerase activity while protease treatment solubilized mtDNA, suggesting the direct interaction of mtDNA with membrane protein(s). The use of a novel approach to detect mtDNA fragments specifically retained by the mitochondrial membranes after Sal I digestion of the complex suggests that specific mtDNA sequences anchor mtDNA to mitochondrial membranes.
Plant Mol Biol 1991 Feb
PMID:Isolation from wheat mitochondria of a membrane-associated high molecular weight complex involved in DNA synthesis. 189 1

The epipodophyllotoxin etoposide is an inhibitor of topoisomerase II. The effects of this agent on gene expression, particularly the transcriptional induction of genes implicated in growth control, are unknown. The present results demonstrate that etoposide induces expression of the c-jun protooncogene in HL-60 myeloid leukemia cells. This induction of c-jun expression was maximal at 3 hr and was transient. Similar findings were obtained in the human U-937 myeloid leukemia cell line. Nuclear run-on assays demonstrated that the induction of c-jun expression by etoposide is regulated at the transcriptional level. The results further demonstrate that etoposide-induced c-jun expression occurs in association with the appearance of c-fos transcripts. Moreover, the c-jun gene is induced by etoposide during periods of oligonucleosomal DNA cleavage, which is characteristic of programmed cell death. These findings suggest that transcriptional induction of c-jun expression represents a signaling pathway activated in the cellular response to etoposide-induced DNA damage.
Mol Pharmacol 1991 Jun
PMID:Activation of the c-jun protooncogene in human myeloid leukemia cells treated with etoposide. 190 80

Bacteriophage T4 gene 17 amplification mutants (Hp17) selected by growth of gene 17 amber mutants on ochre suppressor strains of Escherichia coli carry two to more than sixfold tandem head-to-tail repeats of the gene 17-18 region (Wu & Black, 1987). We characterized the structures of Hp17 isolates by restriction enzyme mapping and Southern blot analysis. The left and right boundaries of the amplified sequences were mapped within genes 16 and gene 18 or 19, respectively. The TaqI-restriction fragments containing the novel junctions arising from fusion of the amplified gene were then cloned and sequenced. Three Hp17 mutants arose from rearrangement in one five base-pair (bp) block within a G + C-rich region of partial homology (24 bp with 4 mismatches) between genes 16 and 19. Moreover, an oligonucleotide probe showed that 190/191 mutants isolated had recombined within the 5 bp block, and other rearrangements within this 24 bp region were not detected. Only one anomalous Hp mutant rearranged elsewhere between genes 16 and 18 in a 14 bp homology region with one mismatch. Elimination of gene alt of phage T4 is required for isolation of Hp17 mutants, apparently because more DNA can be packaged into alt- heads. Requirements for the dispensable replication and recombination genes of T4 were probed; T4 topoisomerase (39, 52, 60), primase (58/61), and uvsX are required, whereas the host recA gene and T4 denV gene do not appear to be required for isolation of the Hp17 mutants. The evidence suggests an initiating sequence-specific rearrangement leads to the T4 Hp17 amplification mutants.
J Mol Biol 1991 Apr 20
PMID:Reiterated gene amplifications at specific short homology sequences in phage T4 produce Hp17 mutants. 202 46

A multidrug-resistant variant of the P388 leukemia cell line exhibits multiple biochemical changes, including reduced drug accumulation and markedly reduced DNA strand breakage induced by anthracyclines. To investigate whether the reduced formation of drug-induced DNA breaks was due to alteration of DNA topoisomerase II activity, nuclear extracts and partially purified enzymes from the sensitive line and the resistant subline were compared. DNA topoisomerase II activity in 0.35 M NaCl nuclear extracts from sensitive cells was approximately 1.7 times higher than that found in extracts from resistant cells, as determined by ability to unknot P4 phage DNA. In addition, it was found that teniposide-stimulated topoisomerase II DNA cleavage activity of nuclear extract from resistant cells was at least 10-fold lower than that from sensitive cells. This differential sensitivity paralleled a similar drug response of nuclei, as determined by the alkaline elution method. However, partially purified DNA topoisomerase II showed similar drug sensitivity in both cell lines. This finding suggests the presence of a modulating factor, which may be lost during purification. These results, indicating a reduction of both catalytic activity and DNA cleavage activity of DNA topoisomerase II in P388 multidrug-resistant cells, emphasize the importance of DNa topoisomerase function in the resistance mechanism. Thus, the concomitant involvement of multiple mechanisms could explain the high degree of resistance of these cells.
Mol Pharmacol 1990 Jan
PMID:Evidence of DNA topoisomerase II-dependent mechanisms of multidrug resistance in P388 leukemia cells. 215 5

There is multiple evidence linking the inhibition of DNA topoisomerases I and II with the cytotoxic effects of antitumor drugs such as camptothecin and the DNA intercalators, 4-(9-acridinylamino)methanesulfon-m-anisidine) (mAMSA) and Adriamycin. We have assessed the effect of the DNA intercalator 3-nitrobenzothiazolo(3,2-a)quinolinium (NBQ) on the DNA topoisomerase I and II activities. NBQ has no effect on the activity of purified topoisomerase I, whereas it induced purified topoisomerase II binding to DNA without inducing DNA scission. Above 30 microM, NBQ stimulated formation of double- and single-strand breaks mediated by topoisomerase II in plasmid DNA. Using the alkaline elution technique we determined that NBQ induced single-strand and DNA-protein-associated breaks in the human promyelocytic leukemia cell line HL-60. At sublethal concentrations (less than or equal to 1 microM), NBQ induce HL-60 cells to differentiate. Topoisomerase II-mediated DNA cleavage induced by mAMSA was substantially reduced in NBQ-differentiated cells. Our data suggest that topoisomerase II could play a major role in the biological activity of NBQ in vivo.
Mol Pharmacol 1990 Mar
PMID:Interaction of DNA intercalator 3-nitrobenzothiazolo (3,2-a)quinolinium with DNA topoisomerases: a possible-mechanism for its biological activity. 215 51

A mixed oligonucleotide probe containing sequences encoding a septapeptide found in yeast, Drosophila and human DNA topoisomerase II was used to screen a genomic library of Trypanosoma brucei. A positive was obtained, and nucleotide sequencing shows that the entire gene encoding DNA topoisomerase II of this organism, TbrTOP2, resides within the T. brucei insert of the clone. A single open reading frame of 1221 triplet codons starting from the first ATG was identified; the amino acid sequence deduced from it is highly homologous to other eukaryotic DNA topoisomerase II and corresponds to a 137-kDa polypeptide. Analysis of restriction endonuclease digests of T. brucei DNA by blot hybridization following gel electrophoresis indicates that TbrTOP2 is a single-copy gene.
Mol Biochem Parasitol 1990 Jan 01
PMID:The TOP2 gene of Trypanosoma brucei: a single-copy gene that shares extensive homology with other TOP2 genes encoding eukaryotic DNA topoisomerase II. 215 53

Low concentrations of novobiocin are toxic to permeable yeast cells, but do not inhibit type II topoisomerase activity. Furthermore, the enzyme does not bind specifically to novobiocin-Sepharose. These observations are in agreement with genetical analyses. Mutations at a single locus that confer novobiocin resistance and temperature sensitivity exhibit a similar phenotype to cells treated with novobiocin, but are not topoisomerase II mutants.
Mol Gen Genet 1990 Jan
PMID:The effect of novobiocin on yeast topoisomerase type II. 215 54

Bacteriophage T4 provides a simple model system in which to examine the mechanism of action of antitumor agents that have been proposed to attack type II DNA topoisomerases. Prior results demonstrated that T4 type II DNA topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in phage-infected Escherichia coli: a point mutation in topoisomerase structural gene 39 was shown to confer both m-AMSA-resistant phage growth and m-AMSA-insensitive topoisomerase activity. We report here that a point mutation in T4 topoisomerase structural gene 52 can also independently render both phage growth and topoisomerase activity resistant to m-AMSA. The DNA relaxation and DNA cleavage activities of this newly isolated mutant topoisomerase were significantly insensitive to m-AMSA. The drug-resistance mutation in gene 52, as well as that in gene 39, alters the DNA cleavage site specificity of wild-type T4 topoisomerase. This finding is consistent with a mechanism of drug action in which both topoisomerase and DNA participate in formation of the drug-binding site.
Mol Gen Genet 1990 Mar
PMID:Mutational alteration of the breakage/resealing subunit of bacteriophage T4 DNA topoisomerase confers resistance to antitumor agent m-AMSA. 215 56


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