How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication.
How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication.
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真核生物的回复体如何实现快速有效的DNA复制。
DOI：
10.1016/j.molcel.2016.11.017
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发表时间：
2017-01-05
期刊：
影响因子：
16
通讯作者：
Diffley JFX
中科院分区：
文献类型：
--
作者：
Yeeles JTP;Janska A;Early A;Diffley JFX
真核复制体是一个分子机器，它协调cdc45-mcm-gins(Cmg)复制dna解旋酶与dna聚合酶α，δ、ε和其他蛋白质以1到2kbmin−1的速率复制领先和滞后链模板。我们现在已经用纯化的蛋白质重组了这个复杂的机器，从调控cmg的组装和激活开始。我们表明复制体相关因子MRc1和Csm3/Tof1对体内复制体进展速度至关重要。此外，只有当DNA聚合酶ε与其加工因子增殖细胞核抗原一起催化前链合成时，才会出现最大速率。Dna聚合酶δ可以支持先导链的合成，但速度较慢。DNA聚合酶δ是滞后链合成所必需的，但令人惊讶的是，在DNA聚合酶ε参与之前，它也在建立领先链合成方面发挥了作用。我们认为，在复制应激条件下，这些DNA聚合酶之间的切换也有助于前导链的合成。重建能够在体内复制的真核复制体MRc1直接刺激复制体速率，并在CSM3/TOF1最大速率的辅助下需要由POLε与增殖细胞核抗原一起合成前导链δ通过用纯化的蛋白质重建真核复制体来在建立导链合成中发挥作用，该真核复制体能以体内的速度合成前导和滞后链，Yeeles等人说。揭示了真核复制体快速高效复制DNA的基础。最大速率需要mRc1和csm3/tof1，它们还依赖于在增殖细胞核抗原存在的情况下由POLε合成的前导链。使用这一系统，作者表明，除了在滞后链上发挥作用外，POLδ还可以在移交给POLε之前建立先导链合成方面发挥重要作用。
The eukaryotic replisome is a molecular machine that coordinates the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerases α, δ, and ε and other proteins to copy the leading- and lagging-strand templates at rates between 1 and 2 kb min−1. We have now reconstituted this sophisticated machine with purified proteins, beginning with regulated CMG assembly and activation. We show that replisome-associated factors Mrc1 and Csm3/Tof1 are crucial for in vivo rates of replisome progression. Additionally, maximal rates only occur when DNA polymerase ε catalyzes leading-strand synthesis together with its processivity factor PCNA. DNA polymerase δ can support leading-strand synthesis, but at slower rates. DNA polymerase δ is required for lagging-strand synthesis, but surprisingly also plays a role in establishing leading-strand synthesis, before DNA polymerase ε engagement. We propose that switching between these DNA polymerases also contributes to leading-strand synthesis under conditions of replicative stress. Reconstitution of a eukaryotic replisome capable of in vivo replication rates Mrc1 directly stimulates replisome rate and is aided by Csm3/Tof1 Maximum rates require leading-strand synthesis by Pol ε together with PCNA Pol δ plays a role in the establishment of leading-strand synthesis By reconstituting a eukaryotic replisome with purified proteins that can synthesize both leading and lagging strands at the in vivo rate, Yeeles et al. reveal the basis for rapid and efficient DNA replication by the eukaryotic replisome. Maximum rates require Mrc1 and Csm3/Tof1, and they are also dependent on leading-strand synthesis by Pol ε in the presence of PCNA. Using this system the authors show that, in addition to functioning on the lagging strand, Pol δ can play an important role in establishing leading-strand synthesis before handing over to Pol ε.