Rad50 DNA binding mechanism

Structural mechanism of ATP-dependent DNA binding and DNA end bridging by eukaryotic Rad50

EMBO J. 2016 Apr 1;35(7):759-72.

 

The Mre11-Rad50-Nbs1 (MRN) complex is a central factor in the repair of DNAdouble-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 DNAalong 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 ofABC ATPase domains of the Rad50/SMC/RecN protein family with DNA.

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