DeStefano J J
University of Maryland, College Park 20742.
J Mol Biol. 1994 Nov 4;243(4):558-67. doi: 10.1016/0022-2836(94)90030-2.
The kinetic mechanism of HIV reverse transcriptase catalyzed strand transfer synthesis (i.e. switching of the primer to a new template) from internal regions of natural sequence RNA was investigated. The system consisted of a 142 nucleotide RNA template (donor), primed with a specific 20 nucleotide DNA oligonucleotide that was used to initiate synthesis. An RNA with homology to an internal region of the donor was used as acceptor template. Using 32P-labeled DNA oligonucleotide, the primer-extension products made from full-length synthesis on the donor (108 bases in length) or homologous transfer to and extension on the acceptor (155 bases) were monitored. Results indicated that the maximum efficiency of transfer (the ratio of transfer products to donor-directed+transfer products x 100) in this particular system was about 25% while the theoretical Vmax for the rate of appearance of transfer products at infinite acceptor concentration was about 20-fold lower than the measured rate for full-length donor-directed products. The Km for acceptor template in the transfer reaction was about 8 nM. Experiments using the above donor template hybridized to a specific DNA that has been shown to transfer to the acceptor indicated that RNase H-mediated rapid release of this DNA from the donor while subsequent association with the acceptor was relatively slow.
研究了HIV逆转录酶催化的链转移合成（即将引物切换到新模板）从天然序列RNA内部区域进行时的动力学机制。该系统由一个142个核苷酸的RNA模板（供体）组成，用一个特定的20个核苷酸的DNA寡核苷酸引发合成，该寡核苷酸用于起始合成。与供体内部区域具有同源性的RNA用作受体模板。使用32P标记的DNA寡核苷酸，监测在供体上进行全长合成（长度为108个碱基）或向受体同源转移并在受体上延伸（155个碱基）所产生的引物延伸产物。结果表明，在这个特定系统中，转移的最大效率（转移产物与供体导向产物+转移产物的比率×100）约为25%，而在无限受体浓度下转移产物出现速率的理论Vmax比全长供体导向产物的测量速率低约20倍。转移反应中受体模板的Km约为8 nM。使用上述与已显示可转移到受体的特定DNA杂交的供体模板进行的实验表明，RNase H介导该DNA从供体快速释放，而随后与受体的结合相对较慢。
