Bohlayer William P, DeStefano Jeffrey J
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA.
Biochemistry. 2006 Jun 20;45(24):7628-38. doi: 10.1021/bi051770w.
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.
对HIV逆转录酶（RT）与独特底物的结合进行了测量，这些底物将RNA-DNA或DNA-DNA定位在聚合酶或核糖核酸酶H结构域附近。底物由一个50个核苷酸的模板和长度从23到43个核苷酸的DNA引物组成。使用了五种不同类型的模板链：均一的（1）RNA或（2）DNA，（3）DNA的前20个5'核苷酸和最后的30个RNA，（4）前20个RNA和最后的30个DNA，以及（5）15个DNA核苷酸，接着是5个RNA，然后是30个DNA。设计不同长度的引物以便将RT定位在模板的各个区域上。测定了每种底物的解离速率常数。结果表明，与DNA-DNA相比，与RNA-DNA的结合紧密了几倍，这是由在聚合酶结构域中的结合决定的，并且只需要一个短的5个碱基对的RNA-DNA杂交区域。在聚合酶结构域附近定位有RNA-DNA且在核糖核酸酶H结构域附近定位有DNA-DNA的嵌合底物显示出与完整RNA-DNA底物相当的结合，而那些方向相反的底物则与DNA-DNA相当。有趣的是，第一种构型虽然与RNA-DNA结合得一样紧密，但不能被RT核糖核酸酶H活性切割，这一发现可能在基于核酸的抑制剂开发中得到利用。
