The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK.
The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK.
Cell Rep. 2019 Sep 10;28(11):2777-2783.e4. doi: 10.1016/j.celrep.2019.08.026.
Disassembly of the replisome is the final step of chromosome duplication in eukaryotes. In budding yeast and metazoa, cullin ubiquitin ligases are required to ubiquitylate the Cdc45-MCM-GINS (CMG) helicase that lies at the heart of the replisome, leading to a disassembly reaction that is dependent upon the ATPase known as Cdc48 or p97. Here, we describe the reconstitution of replisome disassembly, using a purified complex of the budding yeast replisome in association with the cullin ligase SCF. Upon addition of E1 and E2 enzymes, together with ubiquitin and ATP, the CMG helicase is ubiquitylated on its Mcm7 subunit. Subsequent addition of Cdc48, together with its cofactors Ufd1-Npl4, drives efficient disassembly of ubiquitylated CMG, thereby recapitulating the steps of replisome disassembly that are observed in vivo. Our findings define the minimal requirements for disassembly of the eukaryotic replisome and provide a model system for studying the disassembly of protein complexes by Cdc48-Ufd1-Npl4.
在真核生物中，复制体的解体是染色体复制的最后一步。在芽殖酵母和后生动物中，需要 cullin 泛素连接酶来泛素化位于复制体核心的 Cdc45-MCM-GINS（CMG）解旋酶，导致依赖于称为 Cdc48 或 p97 的 ATP 酶的解体反应。在这里，我们描述了使用与 cullin 连接酶 SCF 相关的纯化芽殖酵母复制体复合物来重建复制体解体。在添加 E1 和 E2 酶、泛素和 ATP 后，CMG 解旋酶在其 Mcm7 亚基上被泛素化。随后添加 Cdc48 及其辅助因子 Ufd1-Npl4，可有效地驱动泛素化 CMG 的解体，从而再现体内观察到的复制体解体步骤。我们的发现定义了真核复制体解体的最小要求，并提供了一个研究 Cdc48-Ufd1-Npl4 对蛋白质复合物解体的模型系统。
