美国耶鲁大学Franziska Bleichert团队近期取得重要工作进展，他们研究提出了人类DNA复制起始许可的多重机制。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，复制解旋酶的装载是组装DNA复制机器的必要条件。真核生物MCM2-7复制解旋酶马达通过起源识别复合物（ORC）和共装载蛋白作为头对头双六聚体沉积在DNA上，以许可复制起源。尽管在芽殖酵母中进行了广泛的研究，但多细胞真核生物的起源许可机制仍然不明确。

研究人员使用生化重组和电子显微镜来重建人类MCM负载途径。研究人员发现，与酵母不同，ORC的ORC6亚基对人类MCM负载不是必需的，而是增强的。

电子显微镜分析确定了在ORC6存在和不存在的情况下形成MCM双六聚体的几种中间体，包括一种负载DNA的闭环MCM单六聚体中间体，该中间体可以通过多种机制成熟为头对头双六聚物。ORC6和ORC3促进ORC在第一个六聚体的二聚化界面上被募集到MCM-ORC（MO）复合物中，这些复合物与酵母的MO复合物不同，并可能定向ORC用于第二个MCM六聚体的装载。

此外，MCM双六聚体的形成可以通过独立负载的MCM单六聚体二聚来进行，这是由人类MCM2-7六聚体自二聚的倾向所促进的。人类MCM负载的这种灵活性可以提供抵抗细胞复制应激的弹性，重构系统将使未来研究能够解决关于DNA复制起始和复制偶联事件的悬而未决的问题。

附：英文原文

Title: Multiple mechanisms for licensing human replication origins

Author: Yang, Ran, Hunker, Olivia, Wise, Marleigh, Bleichert, Franziska

Issue&Volume: 2024-11-27

Abstract: Loading of replicative helicases is obligatory for the assembly of DNA replication machineries. The eukaryotic MCM2–7 replicative helicase motor is deposited onto DNA by the origin recognition complex (ORC) and co-loader proteins as a head-to-head double hexamer to license replication origins. Although extensively studied in budding yeast1,2,3,4, the mechanisms of origin licensing in multicellular eukaryotes remain poorly defined. Here we use biochemical reconstitution and electron microscopy to reconstruct the human MCM loading pathway. We find that unlike in yeast, the ORC6 subunit of the ORC is not essential for—but enhances—human MCM loading. Electron microscopy analyses identify several intermediates en route to MCM double hexamer formation in the presence and absence of ORC6, including a DNA-loaded, closed-ring MCM single hexamer intermediate that can mature into a head-to-head double hexamer through multiple mechanisms. ORC6 and ORC3 facilitate the recruitment of the ORC to the dimerization interface of the first hexamer into MCM–ORC (MO) complexes that are distinct from the yeast MO complex5,6 and may orient the ORC for second MCM hexamer loading. Additionally, MCM double hexamer formation can proceed through dimerization of independently loaded MCM single hexamers, promoted by a propensity of human MCM2–7 hexamers to self-dimerize. This flexibility in human MCM loading may provide resilience against cellular replication stress, and the reconstitution system will enable studies addressing outstanding questions regarding DNA replication initiation and replication-coupled events in the future.

DOI: 10.1038/s41586-024-08237-8

Source: https://www.nature.com/articles/s41586-024-08237-8