EXTENSION OF MISMATCHED 3' TERMINI OF DNA IS A MAJOR DETERMINANT OF THE INFIDELITY OF HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 REVERSE-TRANSCRIPTASE
EXTENSION OF MISMATCHED 3' TERMINI OF DNA IS A MAJOR DETERMINANT OF THE INFIDELITY OF HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 REVERSE-TRANSCRIPTASE
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DOI：
10.1073/pnas.86.21.8343
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发表时间：
1989-11-01
影响因子：
11.1
通讯作者：
LOEB, LA
中科院分区：
文献类型：
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作者：
PERRINO, FW;PRESTON, BD;LOEB, LA
人类免疫缺陷病毒I型逆转录酶（HIV-1-RT）异常高的错误率表明，这种酶的聚合错误有助于艾滋病病毒的遗传变异。我们通过研究可能导致碱基替换突变的两个不同步骤分析了HIV-1 RT不忠实的机制：核苷酸错误插入和3“-末端DNA错误配对的延伸。我们的研究结果表明，HIV-1逆转录酶的能力，将核苷酸的错配末端是一个主要因素，在生产的突变，这种酶。当通过HIV-1 RT将非互补的dAMP插入模板腺嘌呤的对面时，新生的3“-末端A. C. A错对容易通过随后掺入下一个互补核苷酸而延伸。通过用凝胶电泳测定法定量延伸引物的量，并通过在与噬菌体vphiX 174 DNA中琥珀突变相对的错配引物杂交后测量诱变，来确定核苷酸添加到3 ″-末端A. A、A. C和A.G错配上的频率。错配延伸频率为50-HIV-1 RT比哺乳动物复制酶DNA聚合酶α高1倍。
The unusually high error rate of human immunodeficiency virus type I reverse transcriptase (HIV-1-RT) suggests that polymerization errors by this enzyme contribute to the genetic variability of the AIDS virus. We have analyzed the mechanism for HIV-1 RT infidelity by studying two distinct steps that might lead to base substitution mutations: nucleotide misinsertions and elongation from 3''-terminal DNA mispairs. Our results indicate that the capacity of HIV-1 RT to polymerize nucleotides onto mispaired termini is a major factor in the production of mutations by this enzyme. When a noncomplementary dAMP was inserted opposite a template adenine by HIV-1 RT, the nascent 3''-terminal A.cntdot.A mispair was readily extended by subsequent incorporation of the next complementary nucleotide. The frequencies of nucleotide addition onto 3''-terminal A.cntdot.A, A.cntdot.C, and A.cntdot.G mispairs were determined by quantitating the amount of extended primers with a gel electrophoresis assay and by measuring mutagenesis after hybridization of mismatched primers opposite an amber mutation in bacteriophage .vphi.X174DNA. The mispair extension frequencies are .apprxeq. 50-fold higher by HIV-1 RT than by the mammalian replicative enzyme DNA polymerase .alpha.