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复合体是一种普遍保守的依赖于ATP的传感器和核酸酶，用于DNA断裂，特别是检测和清除被共价结合蛋白阻断的受阻DNA末端。该复合体有能力装载非常多样化的DNA末端，包含自由末端、发夹，但也有大的蛋白质块，并在断裂附近切割DNA。这会产生一个清洁的基板，以供进一步修复。我们最近确定了原核生物Mre11-Rad50与游离DNA末端结合的冷冻EM结构，这让我们第一次深入了解了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”，这是一种大的卷曲状蛋白质环状结构，可以加载到穿过环的DNA末端。在这项提议中，我们想要检验这一假设。冷冻-EM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，以询问依赖于ATP的传感、加载和切割反应，特别是ATP诱导的全球构象控制的机制，瞬时界面开放的性质，DNA上的聚集，以及盘绕环的化学机制。预期的结果将提供一部关于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.