TEMPLATE SWITCHING BY REVERSE-TRANSCRIPTASE DURING DNA-SYNTHESIS
TEMPLATE SWITCHING BY REVERSE-TRANSCRIPTASE DURING DNA-SYNTHESIS
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DOI：
10.1128/jvi.64.9.4321-4328.1990
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发表时间：
1990-09-01
影响因子：
5.4
通讯作者：
TAYLOR, J
中科院分区：
文献类型：
--
作者：
LUO, G;TAYLOR, J
逆转录酶在DNA合成过程中进行模板切换的能力在逆转录病毒基因组复制的模型中是隐含的，在重组和癌基因转导中也是如此。为了理解这种转换，我们使用了纯化的核酸和酶的体外反应。该检测系统包括使用末端标记的DNA引物，以便允许在加入受体模板RNA时，相对于通过模板切换(通过序列同源性)获得的延伸量，定量测定供体模板上的延伸量。我们考察了影响反应效率的几个变量：(I)反应时间，(Ii)受体和供体模板的相对数量，(Iii)供体和受体模板之间序列重叠的程度，以及(Iv)与逆转录酶相关的RNaseH活性的存在或不存在。与RNA模板和正常逆转录酶的基本反应，产生了高达83%的模板切换。在没有RNaseH的情况下，仍然发生了切换，但效率降低了。此外，当供体模板从RNA改变为DNA时，仍然存在切换；毫不奇怪，这在很大程度上不受RNaseH的存在或不存在的影响。最后，研究了RNaseH在初级转录后但在模板切换之前对RNA模板的作用。我们发现，在大多数情况下，原始RNA模板的两端都能够与DNA产物保持联系。这一结果与其他人的工作一致，他们已经证明了RNaseH作为内切酶的作用。
The ability of reverse transcriptase to make template switches during DNA synthesis is implicit in models of retrovirus genome replication, as well as in recombination and oncogene transduction. In order to understand such switching, we used in vitro reactions with purified nucleic acids and enzymes. the assay system involved the use of an end-labeled DNA primer so as to allow the quantitation of elongation on a donor template relative to the amount of elongation achieved by template switching (by means of sequence homology) when an acceptor template RNA was added. We examined several variables that affected the efficiency of the reaction: (i) the reaction time, (ii) the relative amounts of acceptor and donor template, (iii) the extent of sequence overlap between the donor and acceptor templates, and (iv) the presence or absence of RNase H activity associated with the reverse transcriptase. The basic reaction, with RNA templates and normal reverse transcriptase, yielded as much as 83% template switching. In the absence of RNase H, switching still occurred but the efficiency was lowered. Also, when the donor template was changed from RNA to DNA, there was still switching; not surprisingly, this was largely unaffected by the presence or absence of RNase H. Finally, examined the action of the RNase H on RNA templates after primary transcription but prior to template switching. We found that in most cases, both ends of the original RNA template were able to maintain an association with the DNA product. This result was consistent with the work of others who have shown that RNase H acts as an endonuclease.