Structural mechanism of ATP‐dependent DNA binding and DNA end bridging by eukaryotic Rad50. (19th February 2016)

• Record Type: Journal Article
• Authors: Seifert, Florian Ulrich; Lammens, Katja; Stoehr, Gabriele; Kessler, Brigitte; Hopfner, Karl‐Peter
• Is Part Of: EMBO journal. Volume 35:Number 7(2016)
• Page Start: 759
• Page End: 772
• Publication Date: 2016-02-19
• Subjects: DNA repair -- homologous recombination -- microhomology‐mediated end joining -- protein DNA complex -- X‐ray crystallography; Molecular biology -- Periodicals; 572.805
• DOI: 10.15252/embj.201592934
• Languages: English
• ISSNs: 0261-4189

Abstract

Abstract: 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 Ct Mre11 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. Synopsis: 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. Abstract : The structure of the DNA double‐strand break repair protein Rad50 in complex with DNA reveals how ATP‐dependent dimerization of its ABC ATPase domain generates a positively charged platform for binding DNA.