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

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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.