Watson K F, Schendel P L, Rosok M J, Ramsey L R
Biochemistry. 1979 Jul 24;18(15):3210-9. doi: 10.1021/bi00582a004.
A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.
本文描述了一种用于研究RNA指导的双链DNA合成的模型RNA模板-引物系统，该合成由纯化的禽成髓细胞瘤病毒DNA聚合酶及其相关的核糖核酸酶H催化。在互补RNA引物oligo(rI)以及脱氧核苷三磷酸dGTP、dTTP和dATP存在的情况下，3'-(rC)30-40-poly(rA)可指导从RNA模板3'端的特定位点依次合成poly(dT)和poly(dA)。利用该模型RNA模板-引物，本文描述了双链DNA合成的最佳条件。对DNA合成动力学的分析表明，最初poly(dT)合成迅速。经过短暂的时间滞后，poly(dA)合成和与DNA聚合酶相关的核糖核酸酶H活性开始启动。当poly(rA)指导poly(dT)合成时，DNA合成的需求表明新合成的poly(dT)作为poly(dA)合成的模板。此外，使用NaF的选择性抑制剂研究表明，核糖核酸酶H的激活不仅是一个与时间相关的事件，而且是合成DNA反互补链所必需的。为了确定核糖核酸酶H在该合成序列中的具体作用，研究了poly(dA)合成的引物。通过使用甲酰胺-聚丙烯酰胺平板凝胶电泳，结果表明poly(dT)并非同时作为poly(dA)合成的模板和引物，因为在放射性poly(dA)产物中未观察到poly(dT)-poly(dA)共价连接。在用[α-32P]dATP作为底物合成的碱处理poly(dA)产物的纸色谱图上鉴定出2',3'-[32P]AMP，证明了poly(dA)产物中存在rAMP-dAMP磷酸二酯键。因此，在RNA指导的双链DNA合成中，核糖核酸酶H展示了一种新的功能作用。核糖核酸酶H不仅负责在形成poly(rA)-poly(dT)杂交体后降解poly(rA)，而且产生的poly(rA)片段还作为引物用于启动双链DNA第二条链的合成。
