Gerard G F
J Virol. 1981 Feb;37(2):748-54. doi: 10.1128/JVI.37.2.748-754.1981.
The mechanism of action of Moloney murine leukemia virus RNase H III was studied, utilizing the model substrate (A)n. (dT)n and polyacrylamide gel electrophoresis to assay enzyme activity. Examination by electrophoresis on 15% polyacrylamide gels in 7 M urea and on DEAE-cellulose paper in 7 M urea revealed that, early in a reaction with 3Hn. (dT)n as substrate, RNase H III generated products ranging in length from 80 to 90 nucleotides to less than 10 nucleotides and that after extended incubation the limit digest products generated were 3 to 15 nucleotides long. Product oligomers were of the following configuration: 5'-P, 3'-OHn. RNase H III was shown to be an exonuclease requiring free ends in its substrate for activity by the inability to degrade RNA inserted in Escherichia coli ColE1 plasmid DNA. The enzyme was capable of attacking RNA in RNA-DNA hybrids in the 5' to 3' and 3' to 5' directions as demonstrated by the use of 3H, 5'-32P600. (dT)n and cellulose-3Hn. (dT)n. Rnase H III was random in its mode of action because addition of excess unlabeled (A)n. (dT)n to an ongoing reaction with 3Hn. (dT)n as substrate resulted in immediate inhibition of enzyme activity.
利用模型底物（A）n·（dT）n和聚丙烯酰胺凝胶电泳来测定酶活性，对莫洛尼鼠白血病病毒核糖核酸酶H III的作用机制进行了研究。在7M尿素的15%聚丙烯酰胺凝胶上以及在7M尿素的DEAE - 纤维素纸上进行电泳分析表明，在以[3H]（A）n·（dT）n作为底物的反应初期，核糖核酸酶H III产生的产物长度范围从80至90个核苷酸到小于10个核苷酸，并且在长时间孵育后产生的极限消化产物长度为3至15个核苷酸。产物寡聚物具有以下构型：[5'-P，3'-OH]（A）n。核糖核酸酶H III被证明是一种核酸外切酶，由于其无法降解插入大肠杆菌ColE1质粒DNA中的RNA，所以其活性需要底物中有自由末端。通过使用[3H，5'-32P]（A）600·（dT）n和纤维素 - [3H]（A）n·（dT）n证明，该酶能够在5'至3'和3'至5'方向上攻击RNA - DNA杂交体中的RNA。核糖核酸酶H III的作用方式是随机的，因为向正在进行的以[3H]（A）n·（dT）n作为底物的反应中加入过量未标记的（A）n·（dT）n会导致酶活性立即受到抑制。
