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)

YPF1, a heterodimeric protein from Drosophila melanogaster, is a homolog to Ku, the DNA binding subunit of human DNA-dependent protein kinase. This kinase is crucial in transcriptional activation, V(D)J recombination, double-strand break repair, and both topoisomerase and helicase activities. To investigate functional homology between YPF1 and Ku, we examined DNA binding properties of YPF1. Like Ku, at 100 mM KCl, YPF1 binding has no detectable DNA sequence specificity, requires a DNA terminus, and has a concentration-dependent stoichiometry consistent with subsequent translocation along DNA. YPF1 differs from Ku by having a 10(5)-fold higher affinity. At 400 mM KCl, YPF1 still prefers DNA termini but shows binding specificities not observed previously with Ku. In descending order of affinity, YPF1 binds to: specific DNA sequences with a specific polarity and spacing relative to DNA termini; nonspecific linear DNA; and circular DNA. At this higher ionic strength, binding stoichiometry is concentration independent, indicating that YPF1 remains bound to ends. These results demonstrate a strong functional homology between YPF1 and Ku at physiological ionic strength. The strong binding of YPF1 has also allowed us to detect underlying binding specificities that may be specific to YPF1 and its function.
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PMID:DNA binding specificities of YPF1, a Drosophila homolog to the DNA binding subunit of human DNA-dependent protein kinase, Ku. 866 50

Etoposides block cell division by interfering with the action of topoisomerase II, leaving enzyme-DNA double-strand breaks. We found that certain components of the trimeric DNA-dependent protein kinase influence cell survival following etoposide damage. Interestingly, either Ku70- or Ku80-deficient cell lines, but not mutant cell lines of the DNA-PK catalytic sub-unit (DNA-PKcs), were found to be hypersensitive to the effects of etoposide VP16. Ku70- and Ku80-deficient cells can be complemented to an etoposide resistant phenotype by introducing wildtype Ku70 or Ku80 cDNAs. Mutational analysis of introduced Ku70 cDNAs into murine embryonic stem cells deleted for Ku70 (-/-) showed that mutants where heterodimerization and DNA binding functions of Ku were disrupted, also blocked the restoration of etoposide resistance. In contrast with the differential etoposide sensitivity of DNA-PK mutants, both Ku- and DNA-PKcs-deficient cell lines showed G2 ionizing radiation-induced delays, a cell cycle phase where topoisomerase II function is critical. Thus, the topoisomerase II cleaved complexes may be an example of DNA lesions requiring the Ku heterodimer, but not DNA-PK for DNA repair.
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PMID:Differential etoposide sensitivity of cells deficient in the Ku and DNA-PKcs components of the DNA-dependent protein kinase. 966 32

Ku antigen is a heterodimer, comprised of 86- and 70-kDa subunits, which binds preferentially to free DNA ends. Ku is associated with a catalytic subunit of 450 kDa in the DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double-strand break (DSB) repair and V(D)J recombination of immunoglobulin and T-cell receptor genes. We now demonstrate that Ku86 (86-kDa subunit)-deficient Chinese hamster cell lines are hypersensitive to ICRF-193, a DNA topoisomerase II inhibitor that does not produce DSB in DNA. Mutant cells were blocked in G2 at drug doses which had no effect on wild-type cells. Moreover, bypass of this G2 block by caffeine revealed defective chromosome condensation in Ku86-deficient cells. The hypersensitivity of Ku86-deficient cells toward ICRF-193 was not due to impaired in vitro decatenation activity or altered levels of DNA topoisomerase IIalpha or -beta. Rather, wild-type sensitivity was restored by transfection of a Ku86 expression plasmid into mutant cells. In contrast to cells deficient in the Ku86 subunit of DNA-PK, cells deficient in the catalytic subunit of the enzyme neither accumulated in G2/M nor displayed defective chromosome condensation at lower doses of ICRF-193 compared to wild-type cells. Our data suggests a novel role for Ku antigen in the G2 and M phases of the cell cycle, a role that is not related to its role in DNA-PK-dependent DNA repair.
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PMID:Hypersensitivity of Ku-deficient cells toward the DNA topoisomerase II inhibitor ICRF-193 suggests a novel role for Ku antigen during the G2 and M phases of the cell cycle. 974 97

Cells lacking an intact ATM gene are hypersensitive to ionizing radiation and show multiple defects in the cell cycle-coupled checkpoints. DNA damage usually triggers cell cycle arrest through, among other things, the activation of p53. Another DNA-damage responsive factor is NF-kappaB. It is activated by various stress situations, including oxidative stress, and by DNA-damaging compounds such as topoisomerase poisons. We found that cells from Ataxia Telangiectasia patients exhibit a defect in NF-kappaB activation in response to treatment with camptothecin, a topoisomerase I poison. In AT cells, this activation is shortened or suppressed, compared to that observed in normal cells. Ectopic expression of the ATM protein in AT cells increases the activation of NF-kappaB in response to camptothecin. MO59J glioblastoma cells that do not express the DNA-PK catalytic subunit respond normally to camptothecin. These results support the hypothesis that NF-kappaB is a DNA damage-responsive transcription factor and that its activation pathway by DNA damage shares some components with the one leading to p53 activation.
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PMID:The ATM protein is required for sustained activation of NF-kappaB following DNA damage. 1032 72

DNA damage has been documented in neurodegenerative conditions ranging from Alzheimer's disease to stroke. DNA-dependent protein kinase (DNA-PK) is involved in V(D)J recombination and DNA double strand break repair, and may play a role in cell death induced by DNA damage. We now report that cultured hippocampal neurons from severe combined immunodeficient (scid) mice which lack DNA-PK activity are hypersensitive to apoptosis induced by exposure to topoisomerase inhibitors, amyloid beta peptide (A beta) and glutamate. A similar increased vulnerability of hippocampal CA1 and CA3 neurons was observed in adult scid mice after kainate-induced seizures. Our results suggest that DNA-PK activity is important for neuron survival under conditions that may occur in neurological disorders.
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PMID:Hippocampal neurons of mice deficient in DNA-dependent protein kinase exhibit increased vulnerability to DNA damage, oxidative stress and excitotoxicity. 1124 29

Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochrome c from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochrome c upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells.
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PMID:The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c. 1186 76

The ability of cells to rejoin DNA double-strand breaks (DSBs) usually correlates with their radiosensitivity. This correlation has been demonstrated in radiosensitive cells, including the Chinese hamster ovary mutant XRS-5. XRS-5 is defective in a DNA end-binding protein, Ku80, which is a component of a DNA-dependent protein kinase complex used for joining strand breaks. However, Ku80-deficient cells are known to be retarded in cell proliferation and growth as well as other yet to be identified defects. Using custom-made 600-gene cDNA microarray filters, we found differential gene expressions between the wild-type and XRS-5 cells. Defective Ku80 apparently affects the expression of several repair genes, including topoisomerase-I and -IIA, ERCC5, MLH1, and ATM. In contrast, other DNA repair-associated genes, such as GADD45A, EGR1 MDM2 and p53, were not affected. In addition, for large numbers of growth-associated genes, such as cyclins and clks, the growth factors and cytokines were also affected. Down-regulated expression was also found in several categories of seemingly unrelated genes, including apoptosis, angiogenesis, kinase and signaling, phosphatase, stress protein, proto-oncogenes and tumor suppressors, transcription and translation factors. A RT-PCR analysis confirmed that the XRS-5 cells used were defective in Ku80 expression. The diversified groups of genes being affected could mean that Ku80, a multi-functional DNA-binding protein, not only affects DNA repair, but is also involved in transcription regulation. Our data, taken together, indicate that there are specific genes being modulated in Ku80- deficient cells, and that some of the DNA repair pathways and other biological functions are apparently linked, suggesting that a defect in one gene could have global effects on many other processes.
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PMID:Differential gene expression in a DNA double-strand-break repair mutant XRS-5 defective in Ku80: analysis by cDNA microarray. 1195 61

We investigated whether the levels of the DNA-dependent protein kinase (DNA-PK) activity and content correlate with drug sensitivity in different tumour materials and if this can be utilised in predicting treatment outcome. DNA-PK activity and expression were investigated in tumour cells from 8 patients with chronic lymphocytic leukaemia (CLL) and 18 patients with acute myeloid leukaemia (AML), using Western blot and DNA-PK kinase activity assay. Tumour cells from the patients were investigated for their drug sensitivity to topoisomerase II inhibitors (doxorubicin and etoposide), DNA reactive agents (melphalan, 4-hydroxycyclophosphamide and cisplatinum), an antimetabolite (cytosine arabinoside) and an antimicrotubule agent (vincristine) by fluorometric microculture cytotoxicity assay (FMCA). Within each group of leukaemia there was a large variation in both DNA-PK activity and DNA-PKcs expression, while the Ku subunits were expressed more homogeneously. In CLL cells, sensitivity to topoisomerase II inhibitors correlated with DNA-PKcs protein expression (r=0.7174, p=0.0452). In AML samples, sensitivity to DNA cross-linking alkylating agents correlated with Ku86 (r=-0.7512, p=0.0031) and Ku70 (r=-0.6134, p=0.0258) expression. Unexpectedly, DNA-PK activity was found to correlate with sensitivity to vincristine in both CLL (r=0.8557, p=0.0067) and AML (r=0.5480, p=0.0228) cells. The results indicate that DNA-PK is not only involved in the recognition of DNA double-strand breaks (DSB), but also other DNA lesions.
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PMID:DNA-dependent protein kinase in leukaemia cells and correlation with drug sensitivity. 1216 70

Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBA's DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.
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PMID:Ku antigen, an origin-specific binding protein that associates with replication proteins, is required for mammalian DNA replication. 1239 88

Two human small cell lung cancer (SCLC) subpopulations, CPH 54A, and CPH 54B, established from the same patient tumor by in vitro cloning, were investigated. The tumor was classified as intermediate-type SCLC. The cellular sensitivity to ionizing radiation (IR) was previously determined in the two sublines both in vivo and in vitro. Here we measured the etoposide (VP16) sensitivity together with the induction and repair of VP16- and IR-induced DNA double-strand breaks (DSBs). The two subpopulations were found to differ significantly in sensitivity to VP16, with the radioresistant 54B subline also being VP16 resistant. In order to explain the VP16 resistant phenotype several mechanisms where considered. The p53 status, P-glycoprotein, MRP, topoisomerase IIalpha, and Mre11 protein levels, as well as growth kinetics, provided no explanations of the observed VP16 resistance. In contrast, a significant difference in repair of both VP16- and IR-induced DSBs, together with a difference in the levels of the DSB repair proteins DNA-dependent protein kinase (DNA-PK(cs)) and RAD51 was observed. The VP16- and radioresistant 54B subline exhibited a pronounced higher repair rate of DSBs and higher protein levels of both DNA-PK(cs) and RAD51 compared with the sensitive 54A subline. We suggest, that different DSB repair rates among tumor cell subpopulations of individual SCLC tumors may be a major determinant for the variation in clinical treatment effect observed in human SCLC tumors of identical histological subtype.
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PMID:DNA repair rate and etoposide (VP16) resistance of tumor cell subpopulations derived from a single human small cell lung cancer. 1271 Nov 16


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