A mutation in the primer grip region of HIV-1 reverse transcriptase that confers reduced fidelity of DNA synthesis
A mutation in the primer grip region of HIV-1 reverse transcriptase that confers reduced fidelity of DNA synthesis
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DOI：
10.1093/nar/29.24.4963
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发表时间：
2001-12-15
影响因子：
14.9
通讯作者：
Menéndez-Arias, L
中科院分区：
文献类型：
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作者：
Gutiérrez-Rivas, M;Menéndez-Arias, L
人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）引物中的一个补偿性突变（M230I）可以恢复在其RT编码区具有Y115W突变的病毒的复制能力。Y115W的取代损害了DNA聚合酶的活性，产生了一种DNA合成保真度较低的酶。双突变体Y115W/M230I凝胶保真度分析显示，M230I替代增加了突变体Y115W的准确性。Y115W/M230I在DNA/DNA模板检测中显示野生型误插入保真度。然而，当单独存在时，M230I在误插入和错配延伸保真度测定中，以及与三种dNTPs一起进行的引物延伸测定中，具有降低的保真度。突变体M230I与野生型相比，G、C和T对T的误插入效率提高了3.3-16倍。其保真度不受融合位点周围模板/引物核苷酸替换的影响。然而，其准确性明显受到模板链5'悬垂结构的影响。与野生型HIV-1 RT不同，突变体M230I在dT:dG、dT:dC和dT:dT错配时的核苷酸选择性几乎完全取决于正确和错误dNTPs的K-m值，这一特征尚未被描述为其他低保真度的HIV-1 RT突变体。
A compensatory mutation (M230I) in the primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) restores the replication capacity of virus having a Y115W mutation in their RT coding region. The Y115W substitution impairs DNA polymerase activity and produces an enzyme with a lower fidelity of DNA synthesis. Gel-based fidelity assays with the double mutant Y115W/M230I revealed that the M230I substitution increased the accuracy of mutant Y115W. Y115W/M230I showed wild-type misinsertion fidelity in assays performed with DNA/DNA templates. However, when present alone, M230I conferred reduced fidelity as determined in misinsertion and mispair extension fidelity assays, as well as in primer extension assays carried out with three dNTPs. The mutant M230I showed a 3.3-16-fold increase in misinsertion efficiency for G, C and T opposite T, compared with the wild-type enzyme. Its fidelity was not influenced by nucleotide substitutions in the template/primer around the incorporation site. However, its accuracy was apparently affected by the structure of the 5'-overhang of the template strand. Unlike wild-type HIV-1 RT, nucleotide selectivity of mutant M230I at dT:dG, dT:dC and dT:dT mispairs was almost exclusively dependent on the K-m values for correct and incorrect dNTPs, a characteristic that has not been described for other low fidelity mutants of HIV-1 RT.