Tighter binding of HIV reverse transcriptase to RNA-DNA versus DNA-DNA results mostly from interactions in the polymerase domain and requires just a small stretch of RNA-DNA
Tighter binding of HIV reverse transcriptase to RNA-DNA versus DNA-DNA results mostly from interactions in the polymerase domain and requires just a small stretch of RNA-DNA
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DOI：
10.1021/bi051770w
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发表时间：
2006-06-20
期刊：
影响因子：
2.9
通讯作者：
DeStefano, Jeffrey J.
中科院分区：
文献类型：
--
作者：
Bohlayer, William P.;DeStefano, Jeffrey J.
测定了HIV逆转录酶（RT）与位于聚合酶或RNA酶H结构域附近的RNA-DNA或DNA-DNA的独特底物的结合。底物由50个核苷酸的模板和范围从23至43个核苷酸的DNA引物组成。使用五种不同类型的模板链：均质的（1）RNA或（2）DNA，（3）DNA的前20个5'核苷酸和最后30个RNA，（4）前20个RNA和最后30个DNA，以及（5）DNA的15个核苷酸，随后5个RNA，然后30个DNA。设计不同长度的引物以将RT定位在模板的各个区域上。解离速率常数，确定每个基板。结果表明，与RNA-DNA的结合比DNA-DNA的结合紧密几倍是由聚合酶结构域中的结合决定的，并且仅需要短的5个碱基对的RNA-DNA杂交区。具有位于聚合酶结构域附近的RNA-DNA和位于RNA酶H结构域附近的DNA-DNA的嵌合底物显示出与完整RNA-DNA底物相当的结合，而具有相反取向的那些与DNA-DNA相当。有趣的是，第一种构型虽然与RNA-DNA结合紧密，但不能被RT RNA酶H活性切割，这一发现可能用于开发基于核酸的抑制剂。
Binding of HIV reverse transcriptase (RT) to unique substrates that positioned RNA-DNA or DNA-DNA near the polymerase or RNase H domains was measured. The substrates consisted of a 50 nucleotide template and DNA primers ranging from 23 to 43 nucleotides. Five different types of template strands were used: homogeneous (1) RNA or (2) DNA, (3) the first 20 5' nucleotides of DNA and the last 30 RNA, (4) the first 20 RNA and the last 30 DNA, and (5) 15 nucleotides of DNA followed by 5 RNA and then 30 DNA. The different length primers were designed to position RT over various regions of the template. Dissociation rate constants were determined for each of the substrates. Results showed that the severalfold tighter binding to RNA- DNA vs DNA-DNA was determined by binding in the polymerase domain and required only a short 5 base pair RNA-DNA hybrid region. Chimeric substrates with RNA-DNA positioned near the polymerase domain and DNA-DNA near the RNase H domain showed binding comparable to a complete RNA-DNA substrate, while those with the reverse orientation were comparable to DNA-DNA. Interestingly, the first configuration, though binding as tightly as RNA-DNA, could not be cleaved by RT RNase H activity, a finding that could perhaps be exploited in the development of nucleic acid-based inhibitors.