Dynamic copy choice:: Steady state between murine leukemia virus polymerase and polymerase-dependent RNase H activity determines frequency of in vivo template switching
Dynamic copy choice:: Steady state between murine leukemia virus polymerase and polymerase-dependent RNase H activity determines frequency of in vivo template switching
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DOI：
10.1073/pnas.221289898
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发表时间：
2001-10-09
影响因子：
11.1
通讯作者：
Pathak, VK
中科院分区：
文献类型：
--
作者：
Hwang, CK;Svarovskaia, ES;Pathak, VK
我们最近提出了一个逆转录病毒重组的动态复制选择模型，在该模型中，聚合速率和RNA降解速率之间的稳态决定了逆转录酶(RT)模板切换的频率。在体内的逆转录和模板切换过程中，依赖于聚合酶和不依赖于聚合酶的RNase H活性的相对贡献尚未确定。我们建立了一种体内反式互补实验，通过模板切换直接重复缺失，在逆转录病毒载体中重组功能性绿色荧光蛋白基因。缺乏聚合酶和核糖核酸酶H活性的小鼠白血病病毒Gag-Pol蛋白之间的反式互补恢复了病毒的复制。由于在该细胞系中只存在聚合酶非依赖的RNase H活性，因此可以确定聚合酶依赖和非聚合酶依赖的RNase H活性在模板切换中的相对作用。我们还分析了具有聚合酶和核糖核酸酶H突变的双突变，这些突变分别增加和减少了模板切换。双突变体的模板切换频率较低，表明RNaseH突变为显性突变。双突变体与非聚合酶依赖的RNaseH的反式互补不能恢复高的模板切换频率，这表明依赖聚合酶的RNaseH活性是模板切换频率增加所必需的。此外，在存在和不存在羟基脲的情况下，RNaseH突变体的反式互补减慢了逆转录的速度，表明只有在存在聚合酶依赖的RNase H活性的情况下，羟基尿素才增加了模板切换。据我们所知，这是第一次在体内证明聚合酶依赖的RNaseH活性。这些结果为DNA聚合速率和聚合酶依赖的RNaseH活性之间的动态关联提供了强有力的证据，这决定了体内模板切换的频率。
We recently proposed a dynamic copy-choice model for retroviral recombination in which a steady state between the rates of polymerization and RNA degradation determines the frequency of reverse transcriptase (RT) template switching. The relative contributions of polymerase-dependent and polymerase-independent RNase H activities during reverse transcription and template switching in vivo have not been determined. We developed an in vivo trans-complementation assay in which direct repeat deletion through template switching reconstitutes a functional green fluorescent protein gene in a retroviral vector. Complementation in trans between murine leukemia virus Gag-Pol proteins lacking polymerase and RNase H activities restored viral replication. Because only polymerase-independent RNase H activity is present in this cell line, the relative roles of polymerase-dependent and -independent RNase H activities in template switching could be determined. We also analyzed double mutants possessing polymerase and RNase H mutations that increased and decreased template switching, respectively. The double mutants exhibited low template switching frequency, indicating that the RNase H mutations were dominant. Trans-complementation of the double mutants with polymerase-independent RNase H did not restore the high template switching frequency, indicating that polymerase-dependent RNase H activity was essential for the increased frequency of template switching. Additionally, trans-complementation of RNase H mutants in the presence and absence of hydroxyurea, which slows the rate of reverse transcription, showed that hydroxyurea increased template switching only when polymerase-dependent RNase H activity was present. This is, to our knowledge, the first demonstration of polymerase-dependent RNase H activity in vivo. These results provide strong evidence for a dynamic association between the rates of DNA polymerization and polymerase-dependent RNase H activity, which determines the frequency of in vivo template switching.