The structural mechanism of dimeric DONSON in replicative helicase activation
The structural mechanism of dimeric DONSON in replicative helicase activation
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DOI：
10.1016/j.molcel.2023.09.029
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发表时间：
2023-11-16
期刊：
影响因子：
16
通讯作者：
Costa, Alessandro
中科院分区：
文献类型：
--
作者：
Cvetkovic, Milos A.;Passaretti, Paolo;Costa, Alessandro
复制解旋酶的MCM马达在复制起始前作为无活性的双六聚体加载到起始DNA上。激活剂GINS和Cdc 45在S-相变时的募集促进了两种活性CMG解旋酶的合成。虽然酵母的工作建立了起源激活的机制，CMG是如何在高等真核生物中形成的知之甚少。最近已经显示，后生动物下游邻居的儿子（DONSON）提供GINS MCM在CMG组装。这对MCM双六聚体的影响尚不清楚。在这里，我们使用冷冻电子显微镜（cryo-EM）分离的蛋白质复制非洲爪蟾卵提取物，以确定一个双CMG复合物桥由DONSON二聚体。我们发现，拴系元素介导复杂的形成是必不可少的复制。DONSON重组了双CMG中的MCM马达，原始侏儒症患者的突变破坏了DONSON二聚化，影响了人类细胞中的GINS和MCM参与以及非洲爪蟾卵提取物中的DNA合成。
The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the as-sembly of two active CMG helicases. Although work with yeast established the mechanism for origin activa-tion, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins iso-lated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON recon-figures the MCM motors in the double CMG, and primordial dwarfism patients' mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.