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)

The antitumor agent DACA (N-[2-dimethylamino)ethyl]acridine-4-carboxamide) a new DNA intercalating topoisomerase II poison, was distinguishable from clinical topoisomerase poisons (amsacrine, daunorubicin, doxorubicin and etoposide) in its induction of aberrant colonies in the yeast Saccharomyces cerevisiae D5. It was not only more recombinogenic, but was recombinogenic at non-toxic drug concentrations. DACA at 680 microM (2-h exposure time), induced 1.2% aberrant colonies of which 0.32% were mitotic crossing-over events. The presence of the rad52 mutation abolished mitotic crossing-over and greatly increased drug toxicity. The concentration for 50% inhibition of survival of the rad52 mutant was 100 microM, as compared with 4900 microM for the wild-type. Drug toxicity was marginally increased by the presence of rad3 and rad18 mutations. Rad3 mutations increased the incidence of crossing-over events but had little effect on other mutagenic or recombinogenic events. In contrast, the rad18 mutation increased the incidence of all types of aberrant colonies. The inclusion of hydroxyurea and caffeine, as non-specific repair inhibitors, caused weak and strong inhibition, respectively, of all types of aberrant colonies. Inclusion of the protein-synthesis inhibitor cycloheximide reduced mitotic cross-over but had little effect on the incidence of other aberrations. It is concluded that DACA induces lesions which are repaired by a recombinational repair pathway involving the RAD52 product, and that RAD3 and RAD18 products are each involved in the generation of recombinational events.
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PMID:Induction of mitotic crossing-over by the topoisomerase II poison DACA (N-[2-dimethylamino)ethyl]acridine-4-carboxamide) in Saccharomyces cerevisiae. 769 Aug 83

The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability, sgs1 delta strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1 delta mutants, although meiosis I chromosome missegregation has been shown to be elevated sgs1 delta suppresses the slow growth of a top3 delta strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1 delta strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3 delta or a sgs1 delta strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae.
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PMID:SGS1, a homologue of the Bloom's and Werner's syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae. 891 39

A screening procedure which permits identification of compounds based on their activities against specific biological targets directly in a living organism, Saccharomyces cerevisiae, has been established as part of our new drug discovery programme. Use of this assay has provided the first direct evidence that TOP1 and RAD52 proteins are involved in the mode of action of bisdioxopiperazine ICRF compounds, which thus express a mode of action quite distinctive from the other known TOP2 inhibitors evaluated. The functional assay is based on a comparison of pairs of yeast differing in their phenotypes by specific traits: the expression or lack of expression of ectopic human DNA topoisomerase I, with or without that of the RAD52 gene. Amongst a series of anticancer agents, inhibitors of topoisomerase I (camptothecin) were identified as such in yeast expressing human topoisomerase I, whilst the presence or absence of RAD52 protein permitted the discrimination of compounds generating double-stranded DNA breaks, either directly (bleomycin) or involving DNA adduct formation (cisplatin), or indirectly with DNA damage mediated via inhibition of the topoisomerase II enzyme (etoposide). Notably, however, both the RAD52 protein and the lack of TOP1 enzyme appeared implicated in the cytotoxic activities of the series of bisdioxopiperazine ICRF compounds tested. This functional assay in a living organism therefore appears to provide a valuable tool for probing distinctive and specific mode(s) of action of diverse anticancer agents.
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PMID:Differential expression of topoisomerase I and RAD52 protein in yeast reveals new facets of the mechanism of action of bisdioxopiperazine compounds. 1055 49

Bisdioxopiperazines are a unique class of topoisomerase II inhibitors that lock topoisomerase II at a point in the enzyme reaction cycle where the enzyme forms a closed clamp around DNA. We examined cell killing by ICRF-187 and ICRF-193 in yeast cells expressing human topoisomerase II alpha (htop-IIalpha). Expression of htop-IIalpha in yeast cells sensitizes them to both ICRF-187 and ICRF-193, compared with cells expressing yeast topoisomerase II. ICRF-193 is still able to exert growth inhibition in the presence of genes encoding both ICRF-193-resistant and ICRF-193-sensitive htop-IIalpha enzymes, indicating that sensitivity to bisdioxopiperazines is dominant. Killing by ICRF-193 occurs more rapidly, than the killing in yeast cells due to a temperature-sensitive yeast topoisomerase II incubated at the non-permissive temperature. These results are reminiscent of a top-II poison such as etoposide. However, the killing caused by ICRF-193 and ICRF-187 is not enhanced by mutations in the RAD52 pathway. The levels of drug-induced DNA cleavage observed with htop-IIalpha in vitro is insufficient to explain the sensitivity induced by this enzyme in yeast cells. Finally, arrest of cells in G(1) does not protect cells from ICRF-193 lethality, a result inconsistent with killing mechanisms due to catalytic inhibition of top-II or stabilization of a cleavable complex. We suggest that the observed pattern of cell killing is most consistent with a poisoning of htop-II by ICRF-193 by a novel mechanism. The accumulation of closed clamp conformations of htop-II induced by ICRF-193 that are trapped on DNA might interfere with transcription, or other DNA metabolic processes, resulting in cell death.
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PMID:A novel mechanism of cell killing by anti-topoisomerase II bisdioxopiperazines. 1063 19

A mutant allele of SGS1 of Saccharomyces cerevisiae was identified as a suppressor of the slow-growth phenotype of top3 mutants. We previously reported the involvement of Top3 via the interaction with the N-terminal region of Sgs1 in the complementation of methylmethanesulfonate (MMS) sensitivity and the suppression of hyper recombination of a sgs1 mutant. In this study, we found that several amino acids residues in the N-terminal region of Sgs1 between residues 4 and 33 were responsible for binding to Top3 and essential for complementing the sensitivity to MMS of sgsl cells. Two-hybrid assays suggested that the region of Top3 responsible for the binding to Sgs1 was bipartite, with portion in the N- and C-terminal domains. Although disruption of the SGS1 gene suppressed the semi-lethality of the top3 mutant of strain MR, the sgsl-top3 double mutant grew more slowly and was more sensitive to MMS than the sgsl single mutant, indicating that Top3 plays some role independently of Sgs1. The DNA topoisomerase activity of Top3 was required for the Top3 function to repair DNA damages induced by MMS, as shown by the fact that the TOP3 gene carrying a mutation (Phe for Tyr) at the amino acid residue essential for its activity (residue 356) failed to restore the MMS sensitivity of sgs1-top3 to the level of that of the sgs1 single mutant. Epistatic analysis using the sgs1-top3 double mutant, rad52 mutant and sgs1-top3-rad52 triple mutant indicated that TOP3 belongs to the RAD52 recombinational repair pathway.
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PMID:Functional and physical interaction between Sgs1 and Top3 and Sgs1-independent function of Top3 in DNA recombination repair. 1203

Accidental or drug-induced interruption of the breakage and reunion cycle of eukaryotic topoisomerase I (Top1) yields complexes in which the active site tyrosine of the enzyme is covalently linked to the 3' end of broken DNA. The enzyme tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes this protein-DNA link and thus functions in the repair of covalent complexes, but genetic studies in yeast show that alternative pathways of repair exist. Here, we have evaluated candidate genes for enzymes that might act in parallel to Tdp1 so as to generate free ends of DNA. Despite finding that the yeast Apn1 protein has a Tdp1-like biochemical activity, genetic inactivation of all known yeast apurinic endonucleases does not increase the sensitivity of a tdp1 mutant to direct induction of Top1 damage. In contrast, assays of growth in the presence of the Top1 poison camptothecin (CPT) indicate that the structure-specific nucleases dependent on RAD1 and MUS81 can contribute independently of TDP1 to repair, presumably by cutting off a segment of DNA along with the topoisomerase. However, cells in which all three enzymes are genetically inactivated are not as sensitive to the lethal effects of CPT as are cells defective in double-strand break repair. We show that the MRE11 gene is even more critical than the RAD52 gene for double-strand break repair of CPT lesions, and comparison of an mre11 mutant with a tdp1 rad1 mus81 triple mutant demonstrates that other enzymes complementary to Tdp1 remain to be discovered.
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PMID:Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1. 1239 85

DNA topoisomerases play critical roles in a wide range of cellular processes by altering DNA topology to facilitate replication, transcription, and chromosome segregation. Topoisomerases alter DNA topology by introducing transient DNA strand breaks that involve a covalent protein DNA intermediate. Many agents have been found to prevent the religation of DNA strand breaks induced by the enzymes, thereby converting the enzymes into DNA-damaging agents. Repair of the DNA damage induced by topoisomerases is significant in understanding drug resistance arising following treatment with topoisomerase-targeting drugs. We have used the fission yeast Schizosaccharomyces pombe to identify DNA repair pathways that are important for cell survival following drug treatment. S. pombe strains carrying mutations in genes required for homologous recombination such as rad22A or rad32 (homologues of RAD52 and MRE11) are hypersensitive to drugs targeting either topoisomerase I or topoisomerase II. In contrast to results observed with Saccharomyces cerevisiae, S. pombe strains defective in nucleotide excision repair are also hypersensitive to topoisomerase-targeting agents. The loss of DNA replication or DNA damage checkpoints also sensitizes cells to both topoisomerase I and topoisomerase II inhibitors. Finally, repair genes (such as the S. pombe rad8+ gene) with no obvious homologs in other systems also play important roles in causing sensitivity to topoisomerase drugs. Since the pattern of sensitivity is distinct from that seen with other systems (such as the S. cerevisiae system), our results highlight the usefulness of S. pombe in understanding how cells deal with the unique DNA damage induced by topoisomerases.
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PMID:DNA repair functions that control sensitivity to topoisomerase-targeting drugs. 1487 39

RecQ DNA helicases, including yeast Sgs1p and the human Werner and Bloom syndrome proteins, participate in telomere biology, but the underlying mechanisms are not fully understood. Here, we explore the protein sequences and genetic interactors of Sgs1p that function to slow the senescence of telomerase (tlc1) mutants. We find that the S-phase checkpoint function of Sgs1p is dispensable for preventing rapid senescence, but that Sgs1p sequences required for homologous recombination, including the helicase domain and topoisomerase III interaction domain, are essential. sgs1 and rad52 mutations are epistatic during senescence, indicating that Sgs1p participates in a RAD52-dependent recombinational pathway of telomere maintenance. Several mutations that are synthetically lethal with sgs1 mutation and which individually lead to genome instability, including mus81, srs2, rrm3, slx1 and top1, do not speed the senescence of tlc1 mutants, indicating that the rapid senescence of sgs1 tlc1 mutants is not caused by generic genome instability. However, mutations in SLX5 or SLX8, which encode proteins that function together in a complex that is required for viability in sgs1 mutants, do speed the senescence of tlc1 mutants. These observations further define roles for RecQ helicases and related proteins in telomere maintenance.
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PMID:Evidence that the S.cerevisiae Sgs1 protein facilitates recombinational repair of telomeres during senescence. 1642 46

Sesquiterpene lactones (SLs) present a wide range of pharmacological activities. The aim of our study was to investigate the genotoxicity of 15-deoxygoyazensolide using the Salmonella/microsome assay and the yeast Saccharomyces cerevisiae. We also investigated the nature of induced DNA damage using yeast strains defective in DNA repair pathways, such as nucleotide excision repair (RAD3), error prone repair (RAD6), and recombinational repair (RAD52), and in DNA metabolism, such as topoisomerase mutants. 15-deoxygoyasenzolide was not mutagenic in Salmonella typhimurium, but it was mutagenic in S. cerevisiae. The hypersensitivity of the rad52 mutant suggests that recombinational repair is critical for processing lesions resulting from 15-deoxygoyazensolide-induced DNA damage, whereas excision repair and mutagenic systems does not appear to be primarily involved. Top 1 defective yeast strain was highly sensitive to the cytotoxic activity of 15-deoxygoyazensolide, suggesting a possible involvement of this enzyme in the reversion of the putative complex formation between DNA and this SL, possibly due to intercalation. Moreover, the treatment with this lactone caused dose-dependent glutathione depletion, generating pro-oxidant status which facilitates oxidative DNA damage, particularly DNA breaks repaired by the recombinational system ruled by RAD52 in yeast. Consistent with this finding, the absence of Top1 directly affects chromatin remodeling, allowing repair factors to access oxidative damage, which explains the high sensitivity to top1 strain. In summary, the present study shows that 15-deoxygoyazensolide is mutagenic in yeast due to the possible intercalation effect, in addition to the pro-oxidant status that exacerbates oxidative DNA damage.
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PMID:Genotoxicity of 15-deoxygoyazensolide in bacteria and yeast. 1749 14

The resolution of chromosomes during anaphase is a key step in mitosis. Failure to disjoin chromatids compromises the fidelity of chromosome inheritance and generates aneuploidy and chromosome rearrangements, conditions linked to cancer development. Inactivation of topoisomerase II, condensin, or separase leads to gross chromosome nondisjunction. However, the fate of cells when one or a few chromosomes fail to separate has not been determined. Here, we describe a genetic system to induce mitotic progression in the presence of nondisjunction in yeast chromosome XII right arm (cXIIr), which allows the characterisation of the cellular fate of the progeny. Surprisingly, we find that the execution of karyokinesis and cytokinesis is timely and produces severing of cXIIr on or near the repetitive ribosomal gene array. Consequently, one end of the broken chromatid finishes up in each of the new daughter cells, generating a novel type of one-ended double-strand break. Importantly, both daughter cells enter a new cycle and the damage is not detected until the next G2, when cells arrest in a Rad9-dependent manner. Cytologically, we observed the accumulation of damage foci containing RPA/Rad52 proteins but failed to detect Mre11, indicating that cells attempt to repair both chromosome arms through a MRX-independent recombinational pathway. Finally, we analysed several surviving colonies arising after just one cell cycle with cXIIr nondisjunction. We found that aberrant forms of the chromosome were recovered, especially when RAD52 was deleted. Our results demonstrate that, in yeast cells, the Rad9-DNA damage checkpoint plays an important role responding to compromised genome integrity caused by mitotic nondisjunction.
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PMID:Nondisjunction of a single chromosome leads to breakage and activation of DNA damage checkpoint in G2. 2236 15


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