Jacques P S, Wöhrl B M, Ottmann M, Darlix J L, Le Grice S F
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio.
J Biol Chem. 1994 Oct 21;269(42):26472-8.
"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.
利用“BcgI 盒式”诱变技术制备了 p66 人免疫缺陷病毒（HIV）-1 逆转录酶的变体，这些变体在第 224 位谷氨酸残基和第 229 位色氨酸残基之间存在氨基酸替换。突变多肽在体外与野生型 p51 重组，以产生“选择性突变”的异二聚体系列 p66(224A)/p51 - p66(229A)/p51。对纯化的酶进行了二聚化、DNA 聚合酶、核糖核酸酶 H 和 tRNA(Lys-3)结合方面的表征。综合分析表明，虽然 p66 第 224 位谷氨酸残基至第 228 位亮氨酸残基的改变影响极小，但突变体 p66(229A)/p51 的 DNA 聚合酶活性受损。DNase I 足迹分析表明，该突变体与模型模板引物不能形成稳定的复制复合物。体内研究表明，等效突变消除了病毒感染性，这表明色氨酸 229 对 p66 引物握持结构有贡献。
