Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration.
Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration.
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DOI：
10.1038/s41586-022-05121-1
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发表时间：
2022-09
期刊：
影响因子：
64.8
通讯作者：
Marlovits, Thomas C.
中科院分区：
文献类型：
--
作者：
Wald, Jiri;Fahrenkamp, Dirk;Goessweiner-Mohr, Nikolaus;Lugmayr, Wolfgang;Ciccarelli, Luciano;Vesper, Oliver;Marlovits, Thomas C.
霍利迪连接体是所有生命王国 DNA 重组过程中形成的关键中间体。在细菌中，霍利迪连接体由两个同源六聚体 AAA+ ATPase RuvB 马达处理，它们与 RuvA-霍利迪连接体复合物组装在一起，为链交换反应提供能量。尽管它对染色体维护很重要，但该复合物促进分支迁移的结构和机制尚不清楚。在这里，我们使用时间分辨冷冻电子显微镜，获得了 ATP 水解 RuvAB 复合物在七种不同构象状态下的结构，这些结构是在霍利迪连接体的组装和加工过程中捕获的。五种结构共同解析了完整的核苷酸循环，并揭示了 RuvB 中 ATP 水解、核苷酸交换和特定环境构象变化之间的时空关系。由 DNA 脱离的 RuvB 亚基形成的转换器中的协调运动刺激水解和核苷酸交换。转换器的固定使得 RuvB 能够将 ATP 包含的能量转化为杠杆运动，从而产生驱动分支迁移的拉力。我们发现 RuvB 马达与 DNA 底物一起旋转，与不断进行的核苷酸循环一起，形成了通过连续分支迁移进行 DNA 重组的机制基础。我们的数据共同破译了 RuvAB 复合物同源重组的分子原理，阐明了用于六聚体 AAA+ 马达化学机械耦合的离散和连续过渡态中间体，并为针对 AAA+ 马达的状态特异性化合物的设计提供了蓝图。以多种构象捕获的 ATP 水解 RuvAB 复合物的结构为 DNA 重组过程中协调的 ATP 酶和运动活动提供了机制见解。
The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA–Holliday junction complex to energize the strand-exchange reaction. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors. Structures of the ATP-hydrolysing RuvAB complex captured in multiple conformations provide mechanistic insights into coordinated ATPase and motor activity during DNA recombination.
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影响因子：
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