UNEQUAL HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE ERROR RATES WITH RNA AND DNA TEMPLATES
UNEQUAL HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE ERROR RATES WITH RNA AND DNA TEMPLATES
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DOI：
10.1073/pnas.89.15.6919
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发表时间：
1992-08-01
影响因子：
11.1
通讯作者：
KUNKEL, TA
中科院分区：
文献类型：
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作者：
BOYER, JC;BEBENEK, K;KUNKEL, TA
1型人类免疫缺陷病毒（HIV-1）的序列变异部分是由逆转录酶的不准确复制引起的。虽然这种酶在体外与DNA模板合成过程中容易出错，但在病毒生命周期中与负链复制相关的RNA依赖性DNA合成的保真度尚未得到广泛研究。在本研究中，我们已经开发了一个系统，以确定转录和逆转录的保真度，并用它来比较DNA合成的保真度由HIV-1逆转录酶与RNA和DNA模板相同的序列。总的来说，RNA的保真度比DNA高几倍。用两种底物产生的突变体的序列分析显示，对于特定错误，错误率的差异是实质性的。在五个不同的均聚位置处，对于取代和负一个核苷酸错误，与RNA的保真度高>10倍。因为这样的错误可能是由模板-引物滑动引起的，所以该结果表明，与DNA模板-DNA引物相比，RNA模板-DNA引物形成和/或使用错配中间体的频率较低。结果还表明，HIV-1逆转录酶合成与RNA模板DNA引物是错误的，容易在前两个核苷酸的掺入，可能是由于异常的酶-底物相互作用的合成启动。RNA和DNA模板的错误率不相等表明，负链和正链DNA合成过程中的错误可能对HIV-1的突变率没有同等的贡献。这些数据还提供了T7 RNA聚合酶转录过程中取代和移码错误率的估计。
Sequence variation in the type 1 human immunodeficiency virus (HIV-1) results, in part, from inaccurate replication by reverse transcriptase. Although this enzyme is error-prone during synthesis in vitro with DNA templates, the fidelity of RNA-dependent DNA synthesis relevant to minus-strand replication in the virus life cycle has not been examined extensively. In the present study, we have developed a system to determine the fidelity of transcription and reverse transcription and have used it to compare the fidelity of DNA synthesis by the HIV-1 reverse transcriptase with RNA and DNA templates of the same sequence. Overall, fidelity was several-fold higher with RNA than with DNA. Sequence analysis of mutants generated with the two substrates revealed that differences in error rates were substantial for specific errors. Fidelity with RNA was >10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Because such errors likely result from template-primer slippage, this result suggest; that misaligned intermediates are formed and/or used less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 reverse transcriptase synthesis with an RNA template-DNA primer was error-prone during incorporation of the first two nucleotides, perhaps due to aberrant enzyme-substrate interactions as synthesis initiates. The unequal error rates with RNA and DNA templates suggest that mistakes during minus- and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. The data also provide estimates of substitution and frameshift error rates during transcription by T7 RNA polymerase.