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:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ataxia-telangiectasia (A-T) is a recessive human disease characterized by radiation sensitivity, genetic instability, immunodeficiency, and high cancer risk. We previously used expression cloning to identify CAT4.5, a human cDNA that partially suppresses multiple aspects of the A-T phenotype upon transfection into cultured cells. Sequencing CAT4.5 revealed a 1.1-kb intronic fragment followed by a related ORF of 2.5 kb that encodes the near full-length ORF for hTOP3, the first mammalian topoisomerase III to be identified. Endogenous expression of hTOP3 was found in all human tissues tested. Both pCAT4.5 and an antisense hTOP3 construct were able to inhibit spontaneous and radiation-induced apoptosis in A-T fibroblasts, whereas overexpression of a full-length hTOP3 cDNA did not. We postulate that topoisomerase III may be deregulated in A-T cells and that CAT4.5 complements the A-T phenotype via a dominant-negative mechanism. Furthermore, functional correction of hyper-recombination in A-T cells by CAT4.5 supports the hypothesis that the hTOP3 topoisomerase is involved in the control of genomic stability, perhaps in concert with the Bloom or Werner syndrome DNA helicases.
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PMID:Overexpression of a truncated human topoisomerase III partially corrects multiple aspects of the ataxia-telangiectasia phenotype. 911 25

Werner syndrome (WS) is an autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases. The gene responsible for WS encodes a member of the RecQ-like subfamily of DNA helicases. Here we show that its murine homologue maps to murine chromosome 8 in a region syntenic with the human WRN gene. We have deleted a segment of this gene and created Wrn-deficient embryonic stem (ES) cells and WS mice. While displaying reduced embryonic survival, live-born WS mice otherwise appear normal during their first year of life. Nonetheless, although several DNA repair systems are apparently intact in homozygous WS ES cells, such cells display a higher mutation rate and are significantly more sensitive to topoisomerase inhibitors (especially camptothecin) than are wild-type ES cells. Furthermore, mouse embryo fibroblasts derived from homozygous WS embryos show premature loss of proliferative capacity. At the molecular level, wild-type, but not mutant, WS protein copurifies through a series of centrifugation and chromatography steps with a multiprotein DNA replication complex.
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PMID:A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity. 978 47

Mutations in the WRN gene result in Werner syndrome, an autosomal recessive disease in which many characteristics of aging are accelerated. A probable role in some aspect of DNA metabolism is suggested by the primary sequence of the WRN gene product. A recombinant His-tagged WRN protein (WRNp) was overproduced in insect cells using the baculovirus system and purified to near homogeneity by several chromatographic steps. This purification scheme removes both nuclease and topoisomerase contaminants that persist following a single Ni(2+)affinity chromatography step and allows for unambiguous interpretation of WRNp enzymatic activities on DNA substrates. Purified WRNp has DNA-dependent ATPase and helicase activities consistent with its homology to the RecQ subfamily of proteins. The protein also binds with higher affinity to single-stranded DNA than to double-stranded DNA. However, WRNp has no higher affinity for various types of DNA damage, including adducts formed during 4NQO treatment, than for undamaged DNA. Our results confirm that WRNp has a role in DNA metabolism, although this role does not appear to be the specific recognition of damage in DNA.
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PMID:Enzymatic and DNA binding properties of purified WRN protein: high affinity binding to single-stranded DNA but not to DNA damage induced by 4NQO. 1044 47

It has been proposed that cells monitor chromatid catenation status after DNA replication and inhibit progression into mitosis until chromatids are correctly decatenated by topoisomerase II (TopoII). Studies in yeast have suggested that TopoII may interact with RecQ helicases during this process. Using ICRF187, a TopoII catalytic inhibitor that prevents chromatid decatenation without producing DNA strand breaks, we demonstrated that cells deficient of WRN, a human RecQ helicase, displayed a defect in decatenation checkpoint activation, which was corrected by ectopic expression of wild-type WRN. We also provide evidence that BRCA1 is phosphorylated in an ATR-dependent manner in response to decatenation checkpoint activation and that this phosphorylation is not detectable in Werner syndrome cells. Furthermore, ICRF187 treatment resulted in coimmunoprecipitation of WRN and TopoII. Finally, we demonstrated that override of the decatenation checkpoint resulted in enhanced chromosomal damage and apoptosis only in the absence of WRN, but not in normal cells. Our findings suggest that WRN plays a role in the activation of G(2) decatenation checkpoint and that the abortive function of this pathway itself does not appear to be sufficient to cause genomic instability but rather predisposes to genomic instability and apoptotic cell death in the absence of other "caretaker" genes, such as WRN.
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PMID:The G2-phase decatenation checkpoint is defective in Werner syndrome cells. 1281 Jun 61

We report a novel nucleolar interaction between the AAA ATPase p97/VCP and the Werner protein (WRNp), a member of the RecQ helicase family. p97/VCP mediates several important cellular functions in eucaryotic cells, including membrane fusion of the endoplasmic reticulum and Golgi and ubiquitin-dependent protein degradation. Mutations in the WRN gene cause Werner syndrome, a genetic disorder characterized by premature onset of aging symptoms, a higher incidence of cancer, and a high susceptibility to DNA damage caused by topoisomerase inhibitors. We observed that both WRNp and valosin-containing protein (VCP) were present in the nucleoplasm and in nucleolar foci in mammalian cells and that WRNp and p97/VCP physically interacted in the nucleoli. Importantly, the nucleolar WRNp/VCP complex was dissociated by treatment with camptothecin, an inhibitor of topoisomerase I, whereas other WRNp-associated protein complexes, such as WRNp/Ku 80, were not dissociated by this drug. Because WRN syndrome cells are sensitive to topoisomerase inhibitors, these observations suggest that the VCP/WRNp interaction plays an important role in WRN biology. We propose a novel role for VCP in the DNA damage response pathway through modulation of WRNp availability.
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PMID:DNA damage modulates nucleolar interaction of the Werner protein with the AAA ATPase p97/VCP. 1293 74

Werner syndrome (WS) is an inherited genetic disease in which individuals display the premature aging of a selected subset of tissues. The disorder results from the loss of function mutations in the wrn gene. Wrn codes for a member of the RecQ helicase family with a unique nuclease domain. There is significant evidence that the role of wrn is to assist in the repair and reinitiation of DNA replication forks that have stalled. Loss of the wrn helicase imposes a distinct set of phenotypes at the cellular level. These include premature replicative senescence (in a subset of cell types), chromosomal instability, a distinct mutator phenotype, and hypersensitivity to a limited number of DNA damaging agents. Unfortunately, most of these phenotypes are not suitable for the rapid assessment of loss of function of the wrn gene product. However, WS cells have been reported to show abnormal sensitivity to the drug camptothecin (an inhibitor of topoisomerase type I). A rapid assay for this sensitivity would be a useful marker of loss of wrn function. The COMET (single-cell gel electrophoresis) assay is a rapid, sensitive, versatile, and robust technique for the quantitative assessment of DNA damage in eukaryotic cells. Using this assay, we have found that a significantly increased level of strand breaks can be demonstrated in WS cells treated with camptothecin compared with normal controls.
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PMID:Camptothecin sensitivity in Werner syndrome fibroblasts as assessed by the COMET technique. 1524 24

YB-1 is a multifunctional protein involved in the regulation of transcription, translation, and mRNA splicing. In recent years, several laboratories have demonstrated that YB-1 is also directly involved in the cellular response to genotoxic stress. Accordingly, one report has indicated that the Werner syndrome gene product (WRN) is eluted from an YB-1 affinity chromatography column. Werner syndrome is a rare disorder characterized by the premature onset of a number of age-related diseases, including cancer. The gene responsible for Werner syndrome encodes a DNA helicase/exonuclease protein believed to be involved in some aspect of DNA repair with p53. In this study, we demonstrate that the tumor suppressor, p53, bridges the WRN and YB-1 proteins in vitro. Microscopic analyses of fluorescent-tagged proteins and co-immunoprecipitation experiments confirmed the formation of an YB-1/p53/WRN complex in human cells, but only after treatment with UV light. We also confirmed that p53 is a major player in the translocation of GFP-YB-1 fusion proteins from the cytoplasm to several nuclear foci containing WRN proteins upon UV irradiation. Such translocation did not occur in cells treated with the topoisomerase inhibitor, etoposide, or the radiomimetic drug, bleomycin. Such results suggest that an YB-1/p53/WRN complex is formed in response to the emergence of specific DNA lesions in cells.
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PMID:Formation of a nuclear complex containing the p53 tumor suppressor, YB-1, and the Werner syndrome gene product in cells treated with UV light. 1658 8

Werner syndrome (WS) is an inherited disorder characterized by premature onset of aging, genomic instability, and increased cancer incidence. The disease is caused by loss of function mutations of the WRN gene, a RecQ family member with both helicase and exonuclease activities. However, despite its putative tumor-suppressor function, little is known about the contribution of WRN to human sporadic malignancies. Here, we report that WRN function is abrogated in human cancer cells by transcriptional silencing associated with CpG island-promoter hypermethylation. We also show that, at the biochemical and cellular levels, the epigenetic inactivation of WRN leads to the loss of WRN-associated exonuclease activity and increased chromosomal instability and apoptosis induced by topoisomerase inhibitors. The described phenotype is reversed by the use of a DNA-demethylating agent or by the reintroduction of WRN into cancer cells displaying methylation-dependent silencing of WRN. Furthermore, the restoration of WRN expression induces tumor-suppressor-like features, such as reduced colony formation density and inhibition of tumor growth in nude mouse xenograft models. Screening a large collection of human primary tumors (n = 630) from different cell types revealed that WRN CpG island hypermethylation was a common event in epithelial and mesenchymal tumorigenesis. Most importantly, WRN hypermethylation in colorectal tumors was a predictor of good clinical response to the camptothecin analogue irinotecan, a topoisomerase inhibitor commonly used in the clinical setting for the treatment of this tumor type. These findings highlight the importance of WRN epigenetic inactivation in human cancer, leading to enhanced chromosomal instability and hypersensitivity to chemotherapeutic drugs.
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PMID:Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer. 1672 99

Exposure of mammalian cells to agents that induce DNA double-strand breaks typically results in both reciprocal and nonreciprocal chromosome translocations. Over the past decade, breakpoint junctions of a significant number of translocations and other genomic rearrangements, both in clinical tumors and in experimental models, have been analyzed at the DNA sequence level. Based on these data, reasonable inferences regarding the biochemical mechanisms involved in translocations can be drawn. In a few cases, breakpoints have been shown to correlate with sites of double-strand cleavage by agents to which the cells or patients have been exposed, including exogenous rare-cutting endonucleases, radiomimetic compounds, and topoisomerase inhibitors. These results confirm that translocations primarily reflect misjoining of the exchanged ends of two or more double-strand breaks. Many junctions show significant loss of DNA sequence at the breakpoints, suggesting exonucleolytic degradation of DNA ends prior to joining. The size and frequency of these deletions varies widely, both between experimental systems, and among individual events in a single system. Homologous recombination between repetitive DNA sequences does not appear to be a major pathway for translocations associated with double-strand breaks. Rather, the general features of the junction sequences, particularly the high frequency small terminal deletions, the apparent splicing of DNA ends at microhomologies, and gap-filling on aligned double-strand break ends, are consistent with the known biochemical properties of the classical nonhomologous end joining pathway involving DNA-dependent protein kinase, XRCC4 and DNA ligase IV. Nevertheless, cells with deficiencies in this pathway still exhibit translocations, with grossly similar junction sequences, suggesting an alternative but less conservative end joining pathway. Although evidence for participation of specific DNA end processing enzymes in formation of translocations is largely circumstantial, likely candidates include DNA polymerases lambda and mu, Artemis nuclease, polynucleotide kinase/phosphatase, tyrosyl-DNA phosphodiesterase, DNase III, Werner syndrome protein, and the Mre11/Rad50/NBS1 complex.
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PMID:Biochemical mechanisms of chromosomal translocations resulting from DNA double-strand breaks. 1682 25

WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.
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PMID:Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease. 2178 28


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