Morham S G, Shuman S
Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021.
Genes Dev. 1990 Apr;4(4):515-24. doi: 10.1101/gad.4.4.515.
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.
我们已经开发出一种简单有效的遗传筛选方法，用于筛选真核生物DNA拓扑异构酶I的突变等位基因，这些突变等位基因表现出酶功能严重降低或完全丧失。该筛选基于痘苗拓扑异构酶在大肠杆菌溶原菌菌株BL21(DE3)中表达时的极端毒性，其显著特点是易于区分无义突变（导致蛋白质截短）和错义突变。我们观察到，在筛选中鉴定出的100%的候选等位基因最终在DNA水平上都有单碱基变化，导致蛋白质水平上的氨基酸替换，这证明了该方法的有效性。通过用羟胺体外诱变质粒DNA并应用这种表型筛选，我们分离出了五个不同的单氨基酸替换突变体，每个突变体都表现出一种生化表型，即相对于野生型，突变蛋白的特异性DNA松弛活性降低了90%或更多。因此，在拓扑异构酶功能中涉及的氨基酸在酵母和人类拓扑异构酶的同源位置上有相同或相关的对应物。同样的遗传筛选已应用于痘苗拓扑异构酶温度敏感等位基因的选择，从而分离出另外两个单打击突变等位基因，它们在大肠杆菌BL21(DE3)中表现出温度敏感的生长表型。通过拓宽我们的诱变程序，我们期望生成一张痘苗拓扑异构酶功能和一级蛋白质结构的综合图谱，这应该直接适用于真核细胞酶。我们的方法应该适用于筛选其他在细菌中表达有毒的基因中的错义突变和条件突变等位基因。
