CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication
CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication
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DOI：
10.1073/pnas.1418334111
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发表时间：
2014-10-28
影响因子：
11.1
通讯作者：
O'Donnell, Mike E.
中科院分区：
文献类型：
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作者：
Langston, Lance D.;Zhang, Dan;O'Donnell, Mike E.
真核生物中的DNA复制是不对称的，具有单独的DNA聚合酶（Pol）专门用于大量合成前导链和滞后链。Pol α/引发酶在两条链上起始引物，所述两条链在前导链上通过Pol α延伸并且在滞后链上通过Pol δ延伸。CMG（Cdc 45-MCM-GINS）解旋酶围绕前导链，并且被提出用于募集用于前导链合成的Pol β，但是迄今为止CMG和Pol β之间的直接相互作用尚未被证明。在纯化酵母中过表达的CMG解旋酶时，我们检测到CMG和天然Pol β之间的功能复合物。使用纯CMG和Pol β，我们重建了一个稳定的15亚基CMG-Pol β复合物，并表明它是一个功能性聚合酶-解旋酶在体外模型复制叉上。就其本身而言，Pol β的Pol 2催化亚基在CMG依赖性复制中是低效的，但是添加已知结合CMG的Psf 1蛋白亚基的Pol β的Dpb 2蛋白亚基允许与CMG稳定合成。Dpb 2不影响CMG的Pol δ功能，因此我们提出Dpb 2和CMG之间的连接有助于稳定前导链上的Pol δ，作为我们称为CMGE的15亚基前导链全酶的一部分。Pol β和CMG之间的直接结合为Pol β特异性靶向前导链提供了解释，并为真核生物中如何维持链不对称性提供了明确的机制证据。
DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol alpha/primase initiates primers on both strands that are extended by Pol epsilon on the leading strand and by Pol delta on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol epsilon for leading-strand synthesis, but to date a direct interaction between CMG and Pol epsilon has not been demonstrated. While purifying CMG helicase over-expressed in yeast, we detected a functional complex between CMG and native Pol epsilon. Using pure CMG and Pol epsilon, we reconstituted a stable 15-subunitCMG-Pol epsilon complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol epsilon is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol epsilon, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol delta function with CMG, and thus we propose that the connection between Dpb2 andCMGhelps to stabilize Pol epsilon on the leading strand as part of a 15-subunit leading- strand holoenzyme we refer to as CMGE. Direct binding between Pol epsilon and CMG provides an explanation for specific targeting of Pol epsilon to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.