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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]

While recent technological developments contributed to breakthrough advances in single particle cryo-electron microscopy (cryo-EM), sample preparation remains a significant bottleneck for the structure determination of macromolecular complexes. A critical time factor is sample optimization that requires the use of an electron microscope to screen grids prepared under different conditions to achieve the ideal vitreous ice thickness containing the particles. Evaluating sample quality requires access to cryo-electron microscopes and a strong expertise in EM. To facilitate and accelerate the selection procedure of probes suitable for high-resolution cryo-EM, we devised a method to assess the vitreous ice layer thickness of sample coated grids. The experimental setup comprises an optical interferometric microscope equipped with a cryogenic stage and image analysis software based on artificial neural networks (ANN) for an unbiased sample selection. We present and validate this approach for different protein complexes and grid types, and demonstrate its performance for the assessment of ice quality. This technique is moderate in cost and can be easily performed on a laboratory bench. We expect that its throughput and its versatility will contribute to facilitate the sample optimization process for structural biologists. [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]