Rouillon Christophe, Henneke Ghislaine, Flament Didier, Querellou Joël, Raffin Jean-Paul
IFREMER, UMR 6197, Laboratoire de Microbiologie et Environnements Extrêmes, BP 70, F-29280 Plouzané, France.
J Mol Biol. 2007 Jun 1;369(2):343-55. doi: 10.1016/j.jmb.2007.03.054. Epub 2007 Mar 24.
DNA replication in Archaea, as in other organisms, involves large protein complexes called replisomes. In the Euryarchaeota subdomain, only two putative replicases have been identified, and their roles in leading and lagging strand DNA synthesis are still poorly understood. In this study, we focused on the coupling of proliferating cell nuclear antigen (PCNA)-loading mechanisms with DNA polymerase function in the Euryarchaea Pyrococcus abyssi. PCNA spontaneously loaded onto primed DNA, and replication factor C dramatically increased this loading. Surprisingly, the family B DNA polymerase (Pol B) also increased PCNA loading, probably by stabilizing the clamp on primed DNA via an essential motif. In contrast, on an RNA-primed DNA template, the PCNA/Pol B complex was destabilized in the presence of dNTPs, allowing the family D DNA polymerase (Pol D) to perform RNA-primed DNA synthesis. Then, Pol D is displaced by Pol B to perform processive DNA synthesis, at least on the leading strand.
与其他生物体一样，古菌中的DNA复制涉及称为复制体的大型蛋白质复合物。在广古菌亚域中，仅鉴定出两种假定的复制酶，它们在先导链和后随链DNA合成中的作用仍知之甚少。在本研究中，我们重点关注了嗜热栖热菌中增殖细胞核抗原（PCNA）加载机制与DNA聚合酶功能的耦合。PCNA可自发加载到引发的DNA上，复制因子C可显著增加这种加载。令人惊讶的是，B族DNA聚合酶（Pol B）也增加了PCNA的加载，可能是通过一个必需基序稳定引发DNA上的夹子。相反，在RNA引发的DNA模板上，PCNA/Pol B复合物在存在脱氧核苷三磷酸（dNTPs）的情况下会不稳定，从而使D族DNA聚合酶（Pol D）能够进行RNA引发的DNA合成。然后，Pol D被Pol B取代以进行持续的DNA合成，至少在先导链上是这样。
