Shahid Taha, Danazumi Ammar U, Tehseen Muhammad, Alhudhali Lubna, Clark Alice R, Savva Christos G, Hamdan Samir M, De Biasio Alfredo
Bioscience Program, Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
Leicester Institute of Structural and Chemical Biology and Department of Molecular and Cell Biology, University of Leicester, Leicester, UK.
Nature. 2025 May;641(8061):240-249. doi: 10.1038/s41586-025-08766-w. Epub 2025 Mar 19.
Hexameric helicases are nucleotide-driven molecular machines that unwind DNA to initiate replication across all domains of life. Despite decades of intensive study, several critical aspects of their function remain unresolved: the site and mechanism of DNA strand separation, the mechanics of unwinding propagation, and the dynamic relationship between nucleotide hydrolysis and DNA movement. Here, using cryo-electron microscopy (cryo-EM), we show that the simian virus 40 large tumour antigen (LTag) helicase assembles in the form of head-to-head hexamers at replication origins, melting DNA at two symmetrically positioned sites to establish bidirectional replication forks. Through continuous heterogeneity analysis, we characterize the conformational landscape of LTag on forked DNA under catalytic conditions, demonstrating coordinated motions that drive DNA translocation and unwinding. We show that the helicase pulls the tracking strand through DNA-binding loops lining the central channel, while directing the non-tracking strand out of the rear, in a cyclic process. ATP hydrolysis functions as an 'entropy switch', removing blocks to translocation rather than directly powering DNA movement. Our structures show the allosteric couplings between nucleotide turnover and subunit motions that enable DNA unwinding while maintaining dedicated exit paths for the separated strands. These findings provide a comprehensive model for replication fork establishment and progression that extends from viral to eukaryotic systems. More broadly, they introduce fundamental principles of the mechanism by which ATP-dependent enzymes achieve efficient mechanical work through entropy-driven allostery.
六聚体解旋酶是由核苷酸驱动的分子机器，可解开DNA以启动所有生命域中的复制。尽管经过了数十年的深入研究，其功能的几个关键方面仍未得到解决：DNA链分离的位点和机制、解旋传播的力学原理以及核苷酸水解与DNA移动之间的动态关系。在这里，我们使用冷冻电子显微镜（cryo-EM）表明，猿猴病毒40大肿瘤抗原（LTag）解旋酶在复制起点以头对头六聚体的形式组装，在两个对称定位的位点使DNA解链以建立双向复制叉。通过连续异质性分析，我们表征了催化条件下LTag在叉状DNA上的构象景观，证明了驱动DNA易位和解旋的协同运动。我们表明，解旋酶通过中央通道内衬的DNA结合环拉动追踪链，同时在一个循环过程中将非追踪链导向后方。ATP水解起到“熵开关”的作用，消除易位障碍而不是直接为DNA移动提供动力。我们的结构显示了核苷酸周转与亚基运动之间的变构偶联，这使得DNA能够解旋，同时为分离的链保持专用的出口路径。这些发现为从病毒到真核系统的复制叉建立和进展提供了一个全面的模型。更广泛地说，它们引入了ATP依赖酶通过熵驱动的变构实现高效机械功的机制的基本原理。
