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Research Papers

Phenotypic selection and characterization of mutant alleles of a eukaryotic DNA topoisomerase I.

Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021.

Abstract

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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