Fijalkowska I J, Schaaper R M
Laboratory of Molecualr Genetics, NationalInstitute of Enviromental Health Sciences, Research Triangle Park, NC 27709, USA.
Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2856-61. doi: 10.1073/pnas.93.7.2856.
The Escherichia coli dnaQ gene encodes the proofreading 3' exonuclease (epsilon subunit) of DNA polymerase III holoenzyme and is a critical determinant of chromosomal replication fidelity. We constructed by site-specific mutagenesis a mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12-->Ala and Glu-14-->Ala) in the Exo I motif, which, by analogy to other proofreading exonucleases, is essential for the catalytic activity. When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, still binds to the polymerase subunit (alpha subunit or dnaE gene product). When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells became inviable. However, viable dnaQ926 strains could be obtained if they contained one of the dnaE alleles previously characterized in our laboratory as antimutator alleles or if it carried a multicopy plasmid containing the E. coli mutL+ gene. These results suggest that loss of proofreading exonuclease activity in dnaQ926 is lethal due to excessive error rates (error catastrophe). Error catastrophe results from both the loss of proofreading and the subsequent saturation of DNA mismatch repair. The probability of lethality by excessive mutation is supported by calculations estimating the number of inactivating mutations in essential genes per chromosome replication.
大肠杆菌的dnaQ基因编码DNA聚合酶III全酶的校对3'核酸外切酶（ε亚基），是染色体复制保真度的关键决定因素。我们通过定点诱变构建了一个突变体dnaQ926，方法是改变Exo I基序中的两个保守氨基酸残基（Asp-12→Ala和Glu-14→Ala），类似于其他校对核酸外切酶，该基序对催化活性至关重要。当存在于质粒上时，dnaQ926赋予强烈的显性诱变表型，这表明该蛋白虽然缺乏核酸外切酶活性，但仍与聚合酶亚基（α亚基或dnaE基因产物）结合。当将dnaQ926转移到染色体上以取代野生型基因时，细胞变得无法存活。然而，如果dnaQ926菌株含有我们实验室先前鉴定为抗诱变等位基因的dnaE等位基因之一，或者携带含有大肠杆菌mutL+基因的多拷贝质粒，则可以获得存活的dnaQ926菌株。这些结果表明，由于错误率过高（错误灾难），dnaQ926中校对核酸外切酶活性的丧失是致命的。错误灾难是由校对功能的丧失以及随后DNA错配修复的饱和引起的。通过计算估计每条染色体复制中必需基因的失活突变数量，支持了因过度突变导致致死的可能性。
