Uniquely altered DNA replication fidelity conferred by an amino acid change in the nucleotide binding pocket of human immunodeficiency virus type 1 reverse transcriptase

作     者：Lewis, DA Bebenek, K Beard, WA Wilson, SH Kunkel, TA 

作者机构：NIEHS Struct Biol Lab NIH Res Triangle Pk NC 27709 USA 

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

年 卷 期：1999年第274卷第46期

页      面：32924-32930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] 07[理学] 

基　　金：National Institute of Environmental Health Sciences  NIEHS  (Z01ES050161) 

主　　题：碱基配对 碱基序列 结合部位 DNA引物 DNA复制 脱氧核糖核苷酸类/代谢 HIV逆转录酶/遗传学 动力学 模型 分子 分子序列数据 突变 底物特异性 模板 遗传 人类 

摘      要：Arginine 72 in human immunodeficiency virus type I reverse transcriptase (RT), a highly conserved residue among retroviral polymerases and telomerases, forms part of the binding pocket for the nascent base pair. me show here that replacement of Arg(72) by alanine strongly alters fidelity in a highly unusual manner. R72A reverse transcriptase is a frameshift and base substitution anti-mutator polymerase whose increased fidelity results both from increased nucleotide selectivity and from a decreased ability to extend mismatched primer termini. Thus, Arg(72)-substrate interactions in wild-type human immunodeficiency virus type I RT can stabilize incorrect nucleotides allowing misinsertion and promoting extension of mismatched and perhaps misaligned template-primers. In contrast to the higher fidelity at most sites, R72A RT is highly error-prone for misincorporations opposite template T in the sequence context: 5 -C (T) under bar GG. Surprisingly this results mostly from a 1200-fold increase in the apparent K-m for correct dAMP incorporation. Thus, Arg(72) interactions with substrate are critical for the stability of the correct *** base pair when the 5 -C (T) under bar GG sequence is present in the binding pocket for the nascent base pair. Collectively, the data show that a mutant polymerase may yield higher than normal average replication fidelity, yet paradoxically place specific sequences at very high risk of mutation.