Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase H I).
Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase H I).
复制标题
莫洛尼鼠白血病病毒 RNA 指导的 DNA 聚合酶相关 RNase H (RNase H I) 的作用机制。
作者：
G. Gerard
利用模型底物（A）n比较了与Moloney鼠白血病病毒RNA指导的DNA聚合酶（RNase H I）和禽成髓细胞瘤病毒DNA聚合酶的两亚基（α β）形式相关的核糖核酸酶H（RNase H）活性的作用机制。(dT)n和在7 M尿素中的聚丙烯酰胺凝胶电泳以分析消化产物。在25%聚丙烯酰胺凝胶上的检查显示，通过[3 H]（A）（1100）的有限消化产生了较大比例的RNase H I寡核苷酸产物。(dT)n酸不溶性（15-26个核苷酸长）比酸溶性（少于15个核苷酸长）强，而α β RNase H产生的产物则相反。RNA酶H I能够在5'至3'和3'至5'方向上攻击RNA.DNA中的RNA，如通过使用[3 H，3 '-或5'-32P]（A）（380）所证明的。(dT)n和纤维素-[3 H]（A）n。（dT）n. RNA酶H I和α β RNA酶H都降解[3 H]-（A）n。(dT)n基于经典底物竞争实验和[3 H，3 '-或5'-32 P]降解动力学分析，采用部分进行性机制（A）（380）。（dT）n.也就是说，这两种酶通过有限数量的水解事件保持与RNA、DNA底物结合，但在RNA完全降解之前解离。RNase H I和α β RNase H均能降解[3 H]（C）n-[14 C]（A）n-[32 P]（dA）n中的[14 C]（A）n。(dT)n提示逆转录病毒RNA酶H在体外逆转录病毒DNA合成过程中，能在适当的时间去除负链DNA 5'端的tRNA引物。
The mechanism of action of the ribonuclease H (RNase H) activity associated with Moloney murine leukemia virus RNA-directed DNA polymerase (RNase H I) and the two-subunit (alpha beta) form of avian myeloblastosis virus DNA polymerase were compared by utilizing the model substrate (A)n.(dT)n and polyacrylamide gel electrophoresis in 7 M urea to analyze digestion products. Examination on 25% polyacrylamide gels revealed that a larger proportion of the RNase H I oligonucleotide products generated by limited digestion of [3H](A)(1100).(dT)n were acid insoluble (15-26 nucleotides long) than acid soluble (less than 15 nucleotides long), while the opposite was true for products generated by alpha beta RNase H. RNase H I was capable of attacking RNA in RNA.DNA in the 5' to 3' and 3' to 5' directions, as demonstrated by the use of [3H,3'- or 5'-32P](A)(380).(dT)n and cellulose--[3H](A)n.(dT)n. Both RNase H I and alpha beta RNase H degraded [3H]-(A)n.(dT)n with a partially processive mechanism, based upon classical substrate competition experiments and analyses of the kinetics of degradation of [3H,3'- or 5'-32P](A)(380).(dT)n. That is, both enzymes remain bound to a RNA.DNA substrate through a finite number of hydrolytic events but dissociate before the RNA is completely degraded. Both RNase H I and alpha beta RNase H were capable of degrading [14C](A)n in [3H](C)n-[14C](A)n-[32P](dA)n.(dT)n, suggesting that retroviral RNase H is capable of removing the tRNA primer at the 5' terminus of minus strand DNA at the appropriate time during retroviral DNA synthesis in vitro.