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)

Msx2 is a homeodomain transcriptional repressor that exerts tissue-specific actions during craniofacial skeletal and neural development. To identify coregulatory molecules that participate in transcriptional repression by Msx2, we applied a Farwestern expression cloning strategy to identify transcripts encoding proteins that bind Msx2. A lambdagt11 expression library from mouse brain was screened with radiolabeled GST-Msx2 fusion protein encompassing the core suppressor domain of Msx2. A cDNA was isolated that encodes a novel protein fragment that binds radiolabeled Msx2. Homeoprotein binding activity was confirmed by Farwestern analysis of the T7-epitope-tagged recombinant protein fragment, and interactions in vitro require Msx2 residues necessary for transcriptional suppression in vivo. On the basis of biochemical analyses, this novel protein was named MINT, an acronym for Msx2-interacting nuclear target protein. The original clone is part of a 12.6 kb transcript expressed at high levels in testis and at lower levels in calvarial osteoblasts and brain. Multiple clones isolated from a mouse testis library were sequenced to construct a MINT cDNA contig of 11 kb. Starting from an initiator Met in good Kozak context, a large nascent polypeptide of 3576 amino acids is predicted, in contiguous open reading frame with the Msx2 interaction domain residues 2070-2394. Protein sequence analysis reveals that MINT has three N-terminal RNA recognition motifs (RRMs) and four nuclear localization signals. Western blot analysis of fractionated cell extracts reveals that mature approximately 110 kDa (N-terminal) and approximately 250 kDa (C-terminal) MINT protein fragments accumulate in chromatin and nuclear matrix fractions, cosegregating with Msx2 and topoisomerase II. In gel shift assays, the MINT RRM domain selectively binds T- and G-rich DNA sequences; this includes a large G/T-rich inverted repeat element present in the proximal rat osteocalcin (OC) promoter, overlapping three cognates that support OC expression in osteoblasts. MINT and OC mRNAs are reciprocally regulated during differentiation of MC3T3E1 calvarial osteoblasts. Consistent with its proposed role as a nuclear transcriptional factor, transient expression of MINT(1-812) suppresses the FGF/forskolin-activated OC promoter, does not significantly regulate CMV promoter activity, but markedly upregulates the HSV thymidine kinase promoter in MC3T3E1 cells. In toto, these data indicate that the novel nuclear protein MINT binds the homeoprotein Msx2 and coregulates OC during craniofacial development. Msx2 and MINT both target an information-dense, osteoblast-specific regulatory region of the OC proximal promoter, nucleotides -141 to -111. The N-terminal MINT RRM domain represents an authentic dsDNA binding module for this novel vertebrate nuclear matrix protein. Acting as a scaffold protein, MINT potentially exerts both positive and negative regulatory actions by organizing transcriptional complexes in the nuclear matrix.
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PMID:The RRM domain of MINT, a novel Msx2 binding protein, recognizes and regulates the rat osteocalcin promoter. 1045 62

Human topoisomerase II, a nuclear protein involved in chromosome segregation, is the target of amsacrine and other clinically important anticancer drugs. The enzyme is expressed as alpha and beta isoforms whose mutation/down-regulation has been implicated in drug resistance. To understand the role of target mutations in cellular drug resistance, we have used yeast to select and characterize plasmid-borne human topoisomerase IIalpha mutants resistant to amsacrine. Single point changes of Glu571 to Lys (E571K) or Arg486 to Lys (R486K) in the conserved PLRGK motif, both of which reside in the GyrB homology domain of human topoisomerase IIalpha, were frequently selected and could be shown in vivo to confer >25-fold and >100-fold resistance, respectively, to amsacrine and approximately 3-fold cross-resistance to etoposide. Highly purified E571K and R486K human topoisomerase IIalpha proteins required 100-fold higher levels of amsacrine to induce DNA cleavage similar to that of wild-type protein, consistent with a resistance mechanism involving reduced cleavable complex formation. Our functional studies of the R486K mutation, previously identified in two amsacrine-resistant human cell lines and in human biopsy material, establish unequivocally that it confers resistance, and suggest mechanisms for its phenotypic expression in vivo. These results differ significantly from previous work using yeast topoisomerase II as a model system: introduction of the equivalent mutation to R486K (R476K) into the yeast enzyme did not give amsacrine resistance. We conclude that species-specific differences in topoisomerase II enzymes can affect the drug resistance phenotype of particular mutations and highlight the need to study the relevant human homolog.
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PMID:Mutations at arg486 and glu571 in human topoisomerase IIalpha confer resistance to amsacrine: relevance for antitumor drug resistance in human cells. 1072 26

The breakage/reunion reaction of DNA topoisomerase II (TOP2) can be interrupted by DNA intercalators (e.g., doxorubicin), enzyme binders (e.g., etoposide), or DNA lesions (e.g., abasic sites) to produce TOP2-mediated DNA damage. Here, we demonstrate that thiol alkylation of TOP2 can also produce TOP2-mediated DNA damage. This conclusion is supported by the following observations using purified TOP2: (1) Thiol-reactive quinones were shown to induce TOP2-mediated DNA cleavage. (2) Thiol-reactive compounds such as N-ethylmaleimide (NEM), disulfiram, and organic disulfides [e.g., 2,2'-dithiobis(5-nitropyridine)] were also shown to induce TOP2-mediated DNA cleavage with similar reaction characteristics as thiol-reactive quinones. (3) TOP2-mediated DNA cleavage induced by thiol-reactive quinones was completely abolished using mutant yeast TOP2 with all cysteine residues replaced with alanine (cysteineless TOP2). These results suggest the possibility that cellular DNA damage could occur indirectly through thiolation of a nuclear protein, TOP2. The implications of this reaction in carcinogenesis and apoptotic cell death are discussed.
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PMID:Stimulation of topoisomerase II-mediated DNA damage via a mechanism involving protein thiolation. 1125 51

DNA topoisomerase (topo) I is an essential nuclear protein and a target for anticancer drug camptothecin derivatives. As a nuclear protein, topo I is concentrated in the nucleolus. However, this nucleolar distribution of topo I is dynamic. It has been shown recently that topo I rapidly moves out of the nucleolus (nucleolar delocalization) in response to topo I inhibitors. In the present study, we demonstrated that nucleolar delocalization of topo I is associated with its conjugation by SUMOs (small ubiquitin-like modifiers) in response to the topo I inhibitor topotecan. Time-course experiments revealed that SUMO-topo I conjugation occurred at as early as 5 min after drug treatment, which was earlier than its observed nucleolar delocalization. Furthermore, heat shock blocked sumoylation of topo I; it also blocked the nucleolar delocalization of topo I fusion proteins. UBC9 is an E2 (ubiquitin carrier protein)-conjugating enzyme essential for sumoylation. Although overexpression of wild-type UBC9 enhanced both sumoylation and nuclear delocalization of topo I, overexpression of a UBC9 dominant negative mutant attenuated topo I sumoylation and its nucleolar delocalization. Taken together, our results suggest that sumoylation of topo I might serve as an addressing tag for its nucleolar delocalization in response to topo I inhibitors.
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PMID:Nucleolar delocalization of human topoisomerase I in response to topotecan correlates with sumoylation of the protein. 1170 53

DNA topoisomerase II (topo II) is a major nuclear protein that plays an important role in DNA metabolism. We have isolated the gene for topo II ( TOP2) from the filamentous fungus Aspergillus terreus. The deduced amino acid sequence revealed that topo II consists of 1,587 amino acids and has a calculated molecular weight of 180 kDa; the protein expressed in Escherichia coli has an estimated molecular weight of 185 kDa. Expression of topo II polypeptides tagged with yellow fluorescent protein (YFP) in budding yeast suggests that the C-terminal region of the topo II is essential for transport of the fusion protein into the nucleus. The nuclear localization signal (NLS) sequence of topo II is a non-classical bipartite type containing two interdependent, positively charged clusters separated by 15 amino acids. Alanine scanning mutagenesis and deletion analyses showed further that a stretch of 23 amino acid residues (positions 1,234-1,256) is necessary for nuclear import. In addition, we confirmed, using co-immunoprecipitation and two-hybrid analysis, that this non-classical NLS interacts with importin alpha in budding yeast. These results suggest that the fungal topo II NLS is functional in yeast cells.
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PMID:Identification of a single nuclear localization signal in the C-terminal domain of an Aspergillus DNA topoisomerase II. 1243 51

ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa) is a recently identified nuclear protein that binds to one of the inverted CCAAT boxes of the topoisomerase IIalpha (TopoIIalpha) gene promoter. Here, we show that ICBP90 shares structural homology with several other proteins, including Np95, the human and mouse NIRF, suggesting the emergence of a new family of nuclear proteins. Towards elucidating the functions of this family, we analysed the expression of ICBP90 in various cancer or noncancer cell lines and in normal or breast carcinoma tissues. We found that cancer cell lines express higher levels of ICBP90 and TopoIIalpha than noncancer cell lines. By using cell-cycle phase-blocking drugs, we show that in primary cultured human lung fibroblasts, ICBP90 expression peaks at late G1 and during G2/M phases. In contrast, cancer cell lines such as HeLa, Jurkat and A549 show constant ICBP90 expression throughout the entire cell cycle. The effect of overexpression of E2F-1 is more efficient on ICBP90 and TopoIIalpha expression in noncancer cells (IMR90, WI38) than in cancer cells (U2OS, SaOs). Together, these results show that ICBP90 expression is altered in cancer cell lines and is upregulated by E2F-1 overexpression with an efficiency depending on the cancer status of the cell line.
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PMID:ICBP90 belongs to a new family of proteins with an expression that is deregulated in cancer cells. 1283 12

Two distinct types of cell death have been described: apoptosis and necrosis. However, it is becoming increasingly clear that the differences between these two types are far less numerous than initially thought. Morphological analyses might provide important information to distinguish apoptotic from necrotic samples. We recently reported that in necrotic, but not apoptotic, HL-60 human myeloid leukaemia cells, the nuclear protein topoisomerase IIalpha concentrated in nucleoli. In order to ascertain whether or not this phenomenon was restricted to a peculiar cell type or could be detected also in cells of lymphoid lineage, we performed an investigation aimed at defining the localization of topoisomerase IIalpha in apoptotic and necrotic Jurkat human T lymphoblastoid cells. Immunofluorescence staining demonstrated that topoisomerase IIalpha was excluded from the condensed chromatin of apoptotic cells, whereas in necrotic cells it was localized in discrete nuclear dots. Immuno-electron microscopy analysis showed that topoisomerase IIalpha was undetectable in nucleoli of normal and apoptotic cells, whereas it was present in the nucleolus of necrotic cells irrespectively of the type of inducer used (ethanol, H(2)O(2), HgCl(2)). Taken together, our findings identify topoisomerase IIalpha as a potential morphological marker useful to discriminate between apoptotic and necrotic cells.
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PMID:Intranucleolar localization of DNA topoisomerase IIalpha is a distinctive feature of necrotic, but not of apoptotic, Jurkat T-cells. 1450 84

Inverted CCAAT box binding protein of 90kDa (ICBP90) is a nuclear protein involved in the topoisomerase IIalpha (TopoIIalpha) gene expression. It belongs to a family of E3 ligases of the RING finger type and its expression is deregulated in cancer cells. Previous studies have shown that high expression of ICBP90 may impair the control of G1/S transition of the cell cycle in various cancer cell lines. Since PKA signaling pathway is involved in G1/S transition of the cell cycle, the aim of the present study was to investigate whether cAMP signaling pathways involve phosphorylation of ICBP90. Here, we show that phosphorylation of ICBP90 through the cAMP signaling pathway accelerates exit of forskolin-treated cells from the G1 phase and increases binding of ICBP90 to the ICB2 element of the TopoIIalpha gene promoter with a subsequent increase of TopoIIalpha expression. We identify S298 of ICBP90 as target for PKA. We propose that cAMP signaling pathway enhances TopoIIalpha expression through ICBP90 phosphorylation, which may be one of the major events involved in the G1/S transition.
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PMID:Phosphorylation of ICBP90 by protein kinase A enhances topoisomerase IIalpha expression. 1517 47

The precise mechanism of chromosome condensation and decondensation remains a mystery, despite progress over the last 20 years aimed at identifying components essential to the mitotic compaction of the genome. In this study, we analyse the localization and role of the CAP-D2 non-SMC condensin subunit and its effect on the stability of the condensin complex. We demonstrate that a condensin complex exists in Drosophila embryos, containing CAP-D2, the anticipated SMC2 and SMC4 proteins, the CAP-H/Barren and CAP-G (non-SMC) subunits. We show that CAP-D2 is a nuclear protein throughout interphase, increasing in level during S phase, present on chromosome axes in mitosis, and still present on chromosomes as they start to decondense late in mitosis. We analysed the consequences of CAP-D2 loss after dsRNA-mediated interference, and discovered that the protein is essential for chromosome arm and centromere resolution. The loss of CAP-D2 after RNAi has additional downstream consequences on the stability of CAP-H, the localization of DNA topoisomerase II and other condensin subunits, and chromosome segregation. Finally, we discovered that even after interfering with two components important for chromosome architecture (DNA topoisomerase II and condensin), chromosomes were still able to compact, paving the way for the identification of further components or activities required for this essential process.
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PMID:Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids. 1592 65

Nucleolin associates with various DNA repair, recombination, and replication proteins, and possesses DNA helicase, strand annealing, and strand pairing activities. Examination of nuclear protein extracts from human somatic cells revealed that nucleolin and Rad51 co-immunoprecipitate. Furthermore, purified recombinant Rad51 associates with in vitro transcribed and translated nucleolin. Electroporation-mediated introduction of anti-nucleolin antibody resulted in a 10- to 20-fold reduction in intra-plasmid homologous recombination activity in human fibrosarcoma cells. Additionally, introduction of anti-nucleolin antibody sensitized cells to death induced by the topoisomerase II inhibitor, amsacrine. Introduction of anti-Rad51 antibody also reduced intra-plasmid homologous recombination activity and induced hypersensitivity to amsacrine-induced cell death. Co-introduction of anti-nucleolin and anti-Rad51 antibodies did not produce additive effects on homologous recombination or on cellular sensitivity to amsacrine. The association of the two proteins raises the intriguing possibility that nucleolin binding to Rad51 may function to regulate homologous recombinational repair of chromosomal DNA.
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PMID:A novel interaction [corrected] of nucleolin with Rad51. 1660 Jan 79


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