Communication between Mre11 and Rad50 in the MR complex is critical for the sensing, damage signaling, and repair of DNA double-strand breaks. To understand the basis for interregulation between Mre11 and Rad50, we determined the crystal structure of the Mre11-Rad50-ATPgS complex. Mre11 brings the two Rad50 molecules into close proximity and promotes ATPase activity by (1) holding the coiled-coil arm of Rad50 through its C-terminal domain, (2) stabilizing the signature motif and P loop of Rad50 via its capping domain, and (3) forming a dimer through the nuclease domain. ATP-bound Rad50 negatively regulates the nuclease activity of Mre11 by blocking the active site of Mre11. Hydrolysis of ATP disengages Rad50 molecules, and, concomitantly, the flexible linker that connects the C-terminal domain and the capping domain of Mre11 undergoes substantial conformational change to relocate Rad50 and unmask the active site of Mre11. Our structural and biochemical data provide insights into understanding the interplay between Mre11 and Rad50 to facilitate efficient DNA damage repair.

The Rad50 hook interface is crucial for assembly and functions of the Mre11 complex. Previous analyses suggest that Rad50 molecules interact within (intra-complex) or between (inter-complex) dimeric complexes. In this study, we determined the structure of the human Rad50 hook and coiled-coil domain. The data suggest that the predominant structure is the intra-complex, in which the two parallel coiled-coils proximal to the hook form a rod-shape, and that a novel interface within the coiled-coil domains of Rad50 stabilizes the interaction of Rad50 protomers within the dimeric assembly. In yeast, removal of the coiled-coil interface compromised Tel1 activation without affecting DNA repair, while simultaneous disruption of that interface and the hook phenocopied a null mutation. The results demonstrate that the hook and coiled-coil interfaces coordinately promote intra-complex assembly and define it as the functional form of the Mre11 complex.

Mre11 plays an important role in repairing damaged DNA by cleaving broken ends and by providing a platform for other DNA repair proteins. Various Mre11 mutations have been identified in several types of cancer. We have determined the crystal structure of the human Mre11 core (hMre11), which contains the nuclease and capping domains. hMre11 dimerizes through the interfaces between loop β3-α3 from one Mre11 and loop β4-β5 from another Mre11, and between loop α2-β3 from one Mre11 and helices α2 and α3 from another Mre11, and assembles into a completely different dimeric architecture compared with bacterial or archaeal Mre11 homologs. Nbs1 binds to the region containing loop α2-β3 which participates in dimerization. The hMre11 structure in conjunction with biochemical analyses reveals that many tumorigenic mutations are primarily associated with Nbs1 binding and partly with nuclease activities, providing a framework for understanding how mutations inactivate Mre11.

The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in sensing DNA damage, as well as in resecting and tethering DNA ends, and thus participates in double-strand break repair. An earlier structure of Mre11 bound to a short duplex DNA molecule suggested that each Mre11 in a dimer recognizes one DNA duplex to bridge two DNA ends at a short distance. Here, we provide an alternative DNA recognition model based on the structures of Methanococcus jannaschii Mre11 (MjMre11) bound to longer DNA molecules, which may more accurately reflect a broken chromosome. An extended stretch of B-form DNA asymmetrically runs across the whole dimer, with each end of this DNA molecule being recognized by an individual Mre11 monomer. DNA binding induces rigid-body rotation of the Mre11 dimer, which could facilitate melting of the DNA end and its juxtaposition to an active site of Mre11. The identified Mre11 interface binding DNA duplex ends is structurally conserved and shown to functionally contribute to efficient resection, non-homologous end joining, and tolerance to DNA-damaging agents when other resection enzymes are absent. Together, the structural, biochemical, and genetic findings presented here offer new insights into how Mre11 recognizes damaged DNA and facilitates DNA repair.

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