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

DNA双链断裂威胁着所有生命王国的基因组完整性。Mre11-Rad50复合物是一种普遍保守的ATP依赖传感器和DNA断裂核酸酶，特别是用于检测和清洁被共价附着蛋白阻断的DNA末端。这种复合物有能力装载在非常多样化的DNA末端，包括自由末端、发夹，但也包括大的蛋白质块，并在断裂附近切割DNA。这就产生了一个清洁的基底供进一步修复。我们最近确定了与自由DNA末端结合的原核生物Mre11-Rad50的低温电镜结构，这使我们首次了解了其静止和切割状态的结构。我们的分析表明，Mre11-Rad50形成了一种“拓扑特异性核酸酶”，这是一种大的卷曲卷曲的蛋白质环状结构，可以装载到DNA末端，DNA末端通过环。在这个提议中，我们想检验这个假设。低温电镜将与生化分析、协同高速原子力显微镜和蛋白质工程相结合，研究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机制、瞬态界面打开的性质、DNA上的聚类以及卷曲线圈环的化学力学。预期的结果将提供Mre11-Rad50如何检测和处理各种受阻末端的“分子电影”，这是DNA双链断裂修复中神秘的基本机制之一。

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.