Substitutions of Phe61 located in the vicinity of template 5′-overhang influence polymerase fidelity and nucleoside analog sensitivity of HIV-1 reverse transcriptase
Substitutions of Phe61 located in the vicinity of template 5′-overhang influence polymerase fidelity and nucleoside analog sensitivity of HIV-1 reverse transcriptase
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DOI：
10.1074/jbc.m200282200
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发表时间：
2002-06-21
影响因子：
4.8
通讯作者：
Prasad, VR
中科院分区：
文献类型：
--
作者：
Fisher, TS;Prasad, VR
人类免疫缺陷病毒I型逆转录酶（RT）是一种易于出错的DNA聚合酶。它的保真度的结构决定因素是不完全理解。RT/模板引物接触已显示影响其保真度和对核苷类似物抑制剂的敏感性。Phe（61）残基位于指状亚结构域的β 3折叠内，在逆转录病毒RT中高度保守。三元复合物的晶体结构显示Phe（61）接触5 '-模板突出端的第一和第二碱基。为了确定这种接触是否影响dNTP结合口袋，我们在Phe（61）处进行了有限的垂直扫描诱变（Phe-> Ala、Leu、Trp或Tyr）。F61 A突变体显示出最高的保真度增加，其次是F61 L和F61 W变体，其具有中间表型。F61 Y RT的作用最小。F61 A突变体保真度的增加被其正向突变率降低12倍所证实。Phe（61）突变体RT对2 '，3'-二脱氧胸苷三磷酸和2 '，3'-二脱氧氢胸苷三磷酸的敏感性也显示出大幅降低。显示保真度增加最大的突变体（F61 A和F61 L）也是最具抗性的。这些结果表明，人类免疫缺陷病毒1型RT的指状亚结构域和模板5 '-突出端之间的接触是dNTP结合口袋的几何形状的重要决定因素。
Human immunodeficiency virus type I reverse transcriptase (RT) is an error-prone DNA polymerase. Structural determinants of its fidelity are incompletely understood. RT/template primer contacts have been shown to influence its fidelity and sensitivity to nucleoside analog inhibitors. The Phe(61) residue, located within the beta3 sheet of the finger subdomain, is highly conserved among retroviral RTs. The crystal structure of a ternary complex revealed that Phe(61) contacts the first and second bases of the 5'-template overhang. To determine whether such contacts influence the dNTP-binding pocket, we performed a limited vertical scanning mutagenesis (Phe --> Ala, Leu, Trp, or Tyr) at Phe(61). The F61A mutant displayed the highest increase in fidelity, followed by the F61L and F61W variants, which had intermediate phenotypes. F61Y RT had a minimal effect. The increase in fidelity of the F61A mutant was corroborated by a 12-fold decrease in its forward mutation rate. The Phe(61) mutant RTs also displayed large reductions in sensitivity to 2',3'-dideoxythymidine triphosphate and 2',3'-dideohydrothymidine triphosphate. Mutants displaying the largest increase in fidelity (F61A and F61L) were also the most resistant. These results suggest that contacts between the finger subdomain of human immunodeficiency virus type 1 RT and the template 5'-overhang are important determinants of the geometry of the dNTP-binding pocket.