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)

We have developed a simple, effective genetic screen for mutant alleles of eukaryotic DNA topoisomerase I that manifest severely depressed or complete loss of enzymatic function. The screen is based on the extreme toxicity of vaccinia topoisomerase expression in the Escherichia coli lysogen strain BL21(DE3) and is notable for its ease in distinguishing nonsense mutations (that result in truncated proteins) from missense mutations. The power of the method is evinced by our observation that 100% of the candidate alleles identified in the screen were ultimately found to have single-base changes at the DNA level that result in amino acid substitutions at the protein level. By mutagenizing plasmid DNA in vitro with hydroxylamine and applying this phenotypic screen, we have isolated five distinct single amino acid substitution mutants, each of which shows a biochemical phenotype, that is, greater than or equal to 90% reduction in specific DNA relaxing activity of the mutant protein relative to wild type. The amino acids thus implicated in topoisomerase function have identical or related counterparts at homologous positions in the topoisomerases from yeast and man. The same genetic screen has been applied to the selection of temperature-sensitive alleles of the vaccinia topoisomerase, leading to the isolation of two additional single-hit mutant alleles that display a temperature-sensitive growth phenotype in E. coli BL21(DE3). By broadening our mutagenesis procedures, we expect to generate a comprehensive map of vaccinia topoisomerase function and primary protein structure that should have direct application to eukaryotic cellular enzymes. Our methodology should be applicable to the selection of missense and conditional mutant alleles in other genes whose expression in bacteria is toxic.
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PMID:Phenotypic selection and characterization of mutant alleles of a eukaryotic DNA topoisomerase I. 216 40

Site-directed mutagenesis of regions within a plasmid-borne yeast TOP2 gene encoding DNA topoisomerase II and hydroxylamine mutagenesis of the entire plasmid were carried out, and the mutagenized plasmid DNA pools were used separately to transform yeast with a temperature-sensitive top2-4 mutation in the chromosomal TOP2 locus. By selecting transformants that grow in the presence of the antitumor drug amsacrine at 35 degrees C, a nonpermissive temperature for the top2-4 allele, plasmid-borne top2 mutants expressing amsacrine-resistant and physiologically functional DNA topoisomerase II were readily obtained. The causality between amsacrine resistance and the presence of these mutations in yeast DNA topoisomerase II has been firmly established, and this causality in turn shows that, in yeast at least, DNA topoisomerase II is the only significant cellular target of amsacrine. Three classes of such mutants have been identified: one involves single or multiple changes in a sequence PLRGK-MLN located at positions 474-481 of yeast DNA topoisomerase II, a highly conserved motif in all type II DNA topoisomerases; a second involving a single mutation changing Ala642 to threonine or glycine; and a third involving deletions of portions of the carboxy-terminal domain of the yeast enzyme. The nature of drug resistance of these different classes of mutants is discussed. The approaches used in this work should be readily applicable to yeast cells expressing heterologous DNA topoisomerases such as human DNA topoisomerase II alpha. Other DNA topoisomerase II-targeting drugs can also be studied in such a system.
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PMID:Analysis of yeast DNA topoisomerase II mutants resistant to the antitumor drug amsacrine. 813 94

Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects. Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes. Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation. However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity. A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene. Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II. Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action. None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient. We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.
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PMID:Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity. 938 29

Human cells express two isoforms of topoisomerase II, alpha and beta, that are both targeted by anticancer drugs. To investigate acridine resistance mediated by topoisomerase IIbeta, we used a forced molecular evolution approach. A library of mutated topoisomerase IIbeta cDNAs was generated by hydroxylamine mutagenesis and was transformed into the yeast JN394 top2-4. Methyl N-(4'-(9-acridinylamino)-phenyl)carbamate hydrochloride (AMCA) selection identified a resistant transformant able to grow in media containing 76 microg/ml AMCA. Topoisomerase IIbeta with a glutamic acid-to-lysine substitution at position 522 was responsible for the approximately 10-fold resistance to AMCA. The transformant was cross-resistant to methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA) and mAMCA but hypersensitive to etoposide and ellipticine. In vitro, the betaE522K protein was unable to support acridine-stimulated DNA cleavage, suggesting that resistance to these acridines is caused by reduced drug-stimulated DNA cleavage. However, betaE522K showed DNA cleavage with etoposide, and the cleavable complexes formed with etoposide showed greater stability, thus accounting for the hypersensitivity to etoposide. Drug-independent cleavage of an oligonucleotide by betaE522K was reduced compared with the wild-type enzyme. Decatenation and relaxation activities were reduced to 52 and 61% of the wild-type levels, which may explain the slower growth of yeast strain JN394top2-4 expressing betaE522K at the nonpermissive temperature. This study confirms that topoisomerase IIbeta is a target for AMCA and that resistance to AMCA can be mediated by a point mutation at Glu522 in topoisomerase IIbeta. Residue 522 lies within a Rossmann fold in the B' subfragment of topoisomerase II, a region previously implicated in drug interactions.
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PMID:Mutation E522K in human DNA topoisomerase IIbeta confers resistance to methyl N-(4'-(9-acridinylamino)-phenyl)carbamate hydrochloride and methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide but hypersensitivity to etoposide. 1532 34