Structural mechanism of sensing and processing of blocked DNA ends by the DNA double-strand break repair factor Mre11-Rad50

DNA双链断裂修复因子Mre11-Rad50感知和处理封闭DNA末端的结构机制

• 批准号: 505903095
• 负责人: Professor Dr. Karl-Peter Hopfner
• 金额: --
• 依托单位: Genzentrum - Laboratorium für molekulare Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/505903095?language=en
• 关键词: Structural; mechanism; sensing; processing; blocked

DNA double-strand breaks threaten genomic integrity in all kingdoms of life. The Mre11-Rad50 complex is a universally conserved ATP dependent sensor and nuclease for DNA breaks, in particular to detect and clean obstructed DNA ends blocked by covalently attached proteins. The complex has the capability to load on very diverse DNA ends, containing free termini, hairpins, but also large protein blocks, and cut DNA in the vicinity of the break. This generates a clean substrate for further repair. We recently determined the cryo-EM structure of prokaryotic Mre11-Rad50 bound to a free DNA end, which gave a first insight into the resting and cutting state architecture. Our analysis suggest that Mre11-Rad50 forms a “topology-specific nuclease”, a large coiled-coil proteinaceous ring-like structure that can load onto DNA ends, which pass through the ring. In this proposal, we want to test this hypothesis. Cryo-EM will be combined with biochemical assays, collaborative high-speed AFM and protein engineering to interrogate the ATP dependent sensing, loading and cutting reaction, in particular the mechanism of ATP induced global conformational control, the nature of transient interface opening, clustering on DNA, and the chemo-mechanics of the coiled-coil ring. The expected results will provide a “molecular movie” of how Mre11-Rad50 can detect and process diverse obstructed ends, one of the enigmatic, fundamental mechanisms in DNA double-strand break repair.

DNA双链断裂威胁着所有生命王国的基因组完整性。Mre 11-Rad 50复合物是一种普遍保守的ATP依赖性传感器和DNA断裂核酸酶，特别是用于检测和清除被共价连接的蛋白质阻断的受阻DNA末端。该复合物具有装载在非常不同的DNA末端上的能力，所述末端包含游离末端、发夹，但也包含大的蛋白质块，并且在断裂附近切割DNA。这产生了用于进一步修复的干净基板。我们最近确定了原核Mre 11-Rad 50的冷冻电镜结构结合到一个自由的DNA末端，这给了第一个洞察休息和切割状态的架构。我们的分析表明，Mre 11-Rad 50形成一种“拓扑特异性核酸酶”，一种大的卷曲螺旋蛋白质环状结构，可以加载到DNA末端，通过环。在本提案中，我们想检验这一假设。Cryo-EM将与生化分析、协同高速AFM和蛋白质工程相结合，以询问ATP依赖的传感、加载和切割反应，特别是ATP诱导的全局构象控制的机制、瞬时界面开放的性质、DNA上的聚集以及卷曲螺旋环的化学力学。预期的结果将提供一个“分子电影”，Mre 11-Rad 50如何检测和处理不同的受阻末端，这是DNA双链断裂修复中神秘的基本机制之一。