In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways
In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways
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DOI：
10.1042/bj20120959
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发表时间：
2012-10-15
影响因子：
4.1
通讯作者：
Henneke, Ghislaine
中科院分区：
文献类型：
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作者：
Henneke, Ghislaine
使用模型DNA底物和从深海热球菌中纯化的重组蛋白。我已经在体外重组了参与RNA引物消除的酶反应。在我的双标记系统中，聚合酶D进行有效的链置换DNA合成，产生5‘-RNA翻盖，随后由Fen1释放，然后由Lig1连接。在这个途径中，RNase HII的初始切割事件促进了RNA引物对Okazaki片段的去除。此外，我已经证明，聚合酶B能够在5‘端用单一的核糖核苷酸取代下游的DNA链，这是一种由RNase HII在RNA启动子上的一次切割产生的产物。在RNA消除后，聚合酶B的链置换DNA合成和Fen1的翻盖切割相结合的活性为Lig1的连接提供了一个镍底物。Okazaki片段成熟酶和复制DNA聚合酶的独特特性有力地支持了RNA片段拆分中存在两条途径。
Using model DNA substrates and purified recombinant proteins from Pyrococcus abyssi. I have reconstituted the enzymatic reactions involved in RNA primer elimination in vitro. In my dual-labelled system, polymerase D performed efficient strand displacement DNA synthesis, generating 5'-RNA flaps which were subsequently released by Fen1, before ligation by Lig1. In this pathway, the initial cleavage event by RNase HII facilitated RNA primer removal of Okazaki fragments. In addition, I have shown that polymerase B was able to displace downstream DNA strands with a single ribonucleotide at the 5'-end, a product resulting from a single cut in the RNA initiator by RNase HII. After RNA elimination, the combined activities of strand displacement DNA synthesis by polymerase B and flap cleavage by Fen1 provided a nicked substrate for ligation by Lig1. The unique specificities of Okazaki fragment maturation enzymes and replicative DNA polymerases strongly support the existence of two pathways in the resolution of RNA fragments.