英国弗朗西斯克里克研究所Alessandro Costa和John F. X. Diffley合作取得重要工作进展。他们研究利用人类蛋白质可视化MCM双六聚体的加载过程。相关研究成果2024年11月27日在线发表于《自然》杂志上。

据介绍，真核DNA复制始于MCM复制DNA解旋酶，作为DNA复制起点处的头对头双六聚体装载。目前人们对来源识别复合物（ORC）、CDC6和CDT1如何组装双六聚体的理解主要来自芽殖酵母。

研究人员使用生化重建和纯化蛋白质的冷冻电镜，来表征人类双六聚体（hDH）负载。研究人员发现，人类双六聚体与酵母双六聚物（yDH）的DNA结合方式不同，并在六聚体之间的界面上产生大约五个碱基对的未缠绕DNA，如从细胞中分离的hDH所示。

研究人员在hDH组装过程中发现了，与酵母双六聚体在因子募集和依赖性顺序上的几个差异。与酵母不同，ORC的ORC6亚基对于初始MCM募集或hDH装载不是必需的，但有助于形成另一种hDH组装途径，该途径需要ORC1中固有的无序区域，这可能通过MCM-ORC中间体起作用。

总之，这一工作详细介绍了双六聚体，如何在使用序列独立复制起点的生物体中组装，为真核双六聚物组装机制的多样性提供了进一步的证据，并代表了用纯化的人类蛋白质重建DNA复制起始的第一步。

附：英文原文

Title: MCM double hexamer loading visualized with human proteins

Author: Weissmann, Florian, Greiwe, Julia F., Phringer, Thomas, Eastwood, Evelyn L., Couves, Emma C., Miller, Thomas C. R., Diffley, John F. X., Costa, Alessandro

Issue&Volume: 2024-11-27

Abstract: Eukaryotic DNA replication begins with the loading of the MCM replicative DNA helicase as a head-to-head double hexamer at origins of DNA replication1,2,3. Our current understanding of how the double hexamer is assembled by the origin recognition complex (ORC), CDC6 and CDT1 comes mostly from budding yeast. Here we characterize human double hexamer (hDH) loading using biochemical reconstitution and cryo-electron microscopy with purified proteins. We show that the human double hexamer engages DNA differently from the yeast double hexamer (yDH), and generates approximately five base pairs of underwound DNA at the interface between hexamers, as seen in hDH isolated from cells4. We identify several differences from the yeast double hexamer in the order of factor recruitment and dependencies during hDH assembly. Unlike in yeast5,6,7,8, the ORC6 subunit of the ORC is not essential for initial MCM recruitment or hDH loading, but contributes to an alternative hDH assembly pathway that requires an intrinsically disordered region in ORC1, which may work through a MCM–ORC intermediate. Our work presents a detailed view of how double hexamers are assembled in an organism that uses sequence-independent replication origins, provides further evidence for diversity in eukaryotic double hexamer assembly mechanisms9, and represents a first step towards reconstitution of DNA replication initiation with purified human proteins.

DOI: 10.1038/s41586-024-08263-6

Source: https://www.nature.com/articles/s41586-024-08263-6