Artzi H B, Shemesh J, Zeelon E, Amit B, Kleiman L, Gorecki M, Panet A
Bio-Technology General Israel, Ltd., Kiryat Weizmann, Rehovot, Israel.
Arch Biochem Biophys. 1996 Jan 15;325(2):209-16. doi: 10.1006/abbi.1996.0026.
The specificity of the initial cleavage by the RNaseH activity of HIV-1 reverse transcriptase (RT) during minus strong-stop DNA synthesis was studied using the authentic primer/template tRNA(lys)/HIV RNA. We observed that concomitant with the initiation of DNA synthesis, RNaseH activity of HIV RT introduced the first endonucleolytic cuts within the U5 region of the HIV RNA template, mainly 1 and 3 bases away from the primer binding site. To analyze whether the cleavage sites were determined by sequence specificity, the authentic U5 region at one of the cleavage sites was mutated. The change of sequence did not alter the initial cleavage pattern of RNaseH. In order to determine the size of the RNA/DNA hybrid that is required for RNaseH activation during reverse transcription initiation, DNA synthesis was limited by dideoxynucleotides. DNA extension of the tRNA(lys) primer by 17 deoxyribonucleotides but not by 6 deoxyribonucleotides was sufficient to activate the RNaseH site of HIV RT. Taken together, our results indicate that during initiation of minus strongstop DNA synthesis by HIV RT, the first RNaseH-mediated endonucleolytic cut of the genomic RNA is dictated mainly by the length of the nascent DNA and not by sequence preference.
利用真实的引物/模板tRNA(lys)/HIV RNA，研究了HIV-1逆转录酶（RT）在负链强终止DNA合成过程中RNaseH活性的初始切割特异性。我们观察到，随着DNA合成的起始，HIV RT的RNaseH活性在HIV RNA模板的U5区域引入了最初的内切核酸酶切割，主要在距引物结合位点1和3个碱基处。为了分析切割位点是否由序列特异性决定，对其中一个切割位点的真实U5区域进行了突变。序列的改变并没有改变RNaseH的初始切割模式。为了确定逆转录起始过程中激活RNaseH所需的RNA/DNA杂交体的大小，DNA合成受到双脱氧核苷酸的限制。tRNA(lys)引物延伸17个脱氧核糖核苷酸而不是6个脱氧核糖核苷酸足以激活HIV RT的RNaseH位点。综上所述，我们的结果表明，在HIV RT启动负链强终止DNA合成过程中，基因组RNA的第一次RNaseH介导的内切核酸酶切割主要由新生DNA的长度决定，而不是由序列偏好决定。
