Mutations in the primer grip region of HIV reverse transcriptase can increase replication fidelity

作     者：Wisniewski, M Palaniappan, C Fu, ZP Le Grice, SFJ Fay, P Bambara, RA 

作者机构：Univ Rochester Med Ctr Dept Biochem & Biophys Rochester NY 14642 USA Univ Rochester Dept Med Rochester NY 14642 USA Univ Rochester Ctr Canc Rochester NY 14642 USA Case Western Reserve Univ Sch Med Ctr AIDS Res Cleveland OH 44106 USA Case Western Reserve Univ Sch Med Div Infect Dis Cleveland OH 44106 USA 

出 版 物：《JOURNAL OF BIOLOGICAL CHEMISTRY》 (生物化学杂志)

年 卷 期：1999年第274卷第40期

页      面：28175-28184页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 081704[工学-应用化学] 07[理学] 08[工学] 0817[工学-化学工程与技术] 

基　　金：NCI NIH HHS [CA 11198] Funding Source: Medline NIGMS NIH HHS [GM 52263, GM 49573] Funding Source: Medline 

主　　题：碱基错配 DNA引物 DNA复制 HIV逆转录酶/遗传学 诱变 模板 遗传 

摘      要：Mutations in the primer grip region of human immunodeficiency virus reverse transcriptase (HIV-RT) affect its replication fidelity. The primer grip region (residues 227-235) correctly positions the 3 -ends of primers. Point mutations were created by alanine substitution at positions 224-235, Error frequencies were measured by extension of a dG:dA primer-template mismatch. Mutants E224A, P225A, P226A, L228A, and E233A were approximately equal to the wild type in their ability to extend the mismatch. Mutants F227A, W229A, M230A, G231A, and Y232A extended 40, 66, 54, 72, and 76% less efficiently past a dG:dA mismatch compared with the wild type. We also examined the misinsertion rates of dG, dC, or dA across from a DNA template dA using RT mutants F227A and W229A Mutant W229A exhibited high fidelity and did not produce a dC:dA or dC:dA mismatch. Interestingly, mutant F227A displayed high fidelity for dG:dA and dC:dA mismatches but low fidelity for dA:dA misinsertions, This indicates that F227A discriminates against particular base substitutions. However, a primer extension assay with three dNTPs showed that F227A generally displays higher fidelity than the wild type RT. Clearly, primer grip mutations can improve or worsen either the overall or base-specific fidelity of HIV-RT, We hypothesize that wild type RT has evolved to a fidelity that allows genetic variation without compromising yield of viable viruses.