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The Mre11-Rad50-Nbs1 ( MRN) complex is a central factor in the repair of DNA double-strand breaks ( DSBs). The ATP-dependent mechanisms of how MRN detects and endonucleolytically processes DNA ends for the repair by microhomology-mediated end-joining or further resection in homologous recombination are still unclear. Here, we report the crystal structures of the ATPγS-bound dimer of the Rad50 NBD (nucleotide-binding domain) from the thermophilic eukaryote Chaetomium thermophilum ( Ct) in complex with either DNA or CtMre11 RBD (Rad50-binding domain) along with small-angle X-ray scattering and cross-linking studies. The structure and DNA binding motifs were validated by DNA binding experiments in vitro and mutational analyses in Saccharomyces cerevisiae in vivo. Our analyses provide a structural framework for the architecture of the eukaryotic Mre11-Rad50 complex. They show that a Rad50 dimer binds approximately 18 base pairs of DNA along the dimer interface in an ATP-dependent fashion or bridges two DNA ends with a preference for 3′ overhangs. Finally, our results may provide a general framework for the interaction of ABC ATPase domains of the Rad50/ SMC/RecN protein family with DNA. [ABSTRACT FROM AUTHOR]