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DNA double-strand breaks (DSBs) threaten genome stability and are linked to tumorigenesis in humans. Repair of DSBs requires the removal of attached proteins and hairpins through a poorly understood but physiologically critical endonuclease activity by the Mre11-Rad50 complex. Here, we report cryoelectron microscopy (cryo-EM) structures of the bacterial Mre11-Rad50 homolog SbcCD bound to a protein-blocked DNA end and a DNA hairpin. The structures reveal that Mre11-Rad50 bends internal DNA for endonucleolytic cleavage and show how internal DNA, DNA ends, and hairpins are processed through a similar ATP-regulated conformational state. Furthermore, Mre11-Rad50 is loaded onto blocked DNA ends with Mre11 pointing away from the block, explaining the distinct biochemistries of 3′ → 5′ exonucleolytic and endonucleolytic incision through the way Mre11-Rad50 interacts with diverse DNA ends. In summary, our results unify Mre11-Rad50's enigmatic nuclease diversity within a single structural framework and reveal how blocked DNA ends and hairpins are processed. [Display omitted] • Cryo-EM structures of Ec Mre11-Rad50 bound to a protein-blocked DNA end and a hairpin • A single structural framework unifying Mre11-Rad50's enigmatic nuclease diversity • Mre11-Rad50 is loaded onto blocked DNA ends with Mre11 pointing away from the block • Mre11-Rad50 strongly bends internal dsDNA for endonucleolytic cleavage Gut et al. use cryoelectron microscopy and biochemical studies to reveal how the Mre11-Rad50 (MR) nuclease generates an endonucleolytic incision to remove protein blocks from a DNA double-strand break, providing also a unified mechanistic basis for MR's endonuclease, exonuclease, and hairpin-opening activities. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils. • cryo-EM structure of Ec Mre11-Rad50 bound to a DNA break • Mre11 dimer binds the DNA end at the side of Rad50 • Mre11 and Rad50 assemble a transient DNA cutting channel • The coiled coils form a rod-shaped DNA gate and clamp Käshammer et al. use cryoelectron microscopy and biochemical studies to reveal, at near-atomic resolution, how the ATP-dependent nuclease Mre11-Rad50 can sense and process a wide range of DNA termini to enable repair and recombination of breaks and hairpins in chromosomal DNA. [ABSTRACT FROM AUTHOR]