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 Rad50NBD (nucleotide‐binding domain) from the thermophilic eukaryote Chaetomium thermophilum (Ct) in complex with either DNA or CtMre11RBD (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.
See also: H Schüler & C Sjögren (April 2016) and
Y Liu et al (April 2016)
The structure of the eukaryotic DNA double‐strand break repair protein Rad50 in complex with ATPγS and DNA reveals how ATP‐dependent dimerization of Rad50's ABC ATPase domain generates a positively charged platform that binds continuous B‐DNA. The structure provides a framework for the ATP‐dependent interaction of ABC ATPase domains of the Rad50/SMC/RecN family with DNA.
Eukaryotic Mre11 binds to Rad50 via a five‐helix bundle domain.
Rad50:ATPγS binds DNA along the ABC ATPase domain dimer interface.
Rad50's DNA interaction is critical for the repair of topoisomerase‐induced DNA double‐strand breaks.
The Rad50 dimer can bind either continuous B‐DNA or two joined DNA ends.
The EMBO Journal (2016) 35: 759–772
- Received August 27, 2015.
- Revision received December 10, 2015.
- Accepted January 13, 2016.
- © 2016 The Authors
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