Kim B, Hathaway T R, Loeb L A
The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195-3577, USA.
Biochemistry. 1998 Apr 28;37(17):5831-9. doi: 10.1021/bi972672g.
We have used random sequence mutagenesis and complementation in a bacterial selection system to establish a large library of immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with amino acid substitutions in the beta3-beta4 region of the fingers subdomain [Kim, B., Hathaway, T. R., and Loeb, L. A. (1996) J. Biol. Chem. 271, 4872-4878]. We show here that one of these mutants, D76V, exhibits increased accuracy of copying both DNA and RNA templates in a primer extension assay with biased dNTP pools. More detailed analysis of DNA-dependent polymerization showed that the D76V mutation conferred an up to 14-fold increase in fidelity of nucleotide insertion and a 9-fold reduced mutation rate in an M13mp2 lacZalpha forward mutation assay. Substitution at D76 with positively charged (D76R) and nonpolar (D76V and D76I) residues increased replicational accuracy, while substitutions with negatively charged (D76E) and polar residues (D76S and D76C) had little effect on fidelity. We propose that D76 affects replicational accuracy by mediating interaction between the fingers subdomain and the single-stranded template. Our work shows that the Escherichia coli complementation system can yield HIV RT mutants with increased fidelity that have not been isolated from the natural host and that are valuable in understanding the molecular bases of replicational accuracy.
我们利用随机序列诱变技术，并在细菌筛选系统中进行互补，建立了一个庞大的1型免疫缺陷病毒（HIV-1）逆转录酶（RT）突变体文库，这些突变体在指状亚结构域的β3-β4区域存在氨基酸替换[Kim, B., Hathaway, T. R., and Loeb, L. A. (1996) J. Biol. Chem. 271, 4872-4878]。我们在此表明，其中一个突变体D76V，在使用偏向性dNTP库的引物延伸试验中，对DNA和RNA模板的复制准确性有所提高。对依赖DNA的聚合反应进行更详细的分析表明，在M13mp2 lacZα正向突变试验中，D76V突变使核苷酸插入的保真度提高了14倍，突变率降低了9倍。用带正电荷的（D76R）和非极性的（D76V和D76I）残基替换D76可提高复制准确性，而用带负电荷的（D76E）和极性残基（D76S和D76C）替换则对保真度影响不大。我们提出，D76通过介导指状亚结构域与单链模板之间的相互作用来影响复制准确性。我们的工作表明，大肠杆菌互补系统可以产生保真度提高的HIV RT突变体，这些突变体尚未从天然宿主中分离出来，并且对于理解复制准确性的分子基础具有重要价值。
