title: Structural mechanism of ATP-dependent DNA binding and DNA end bridging by eukaryotic Rad50.
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 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 anATP-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.
authors.affiliation: Department of Biochemistry and Gene Center, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Florian Ulrich
authors.initials: FU
authors.lastname: Seifert
authors.affiliation: Department of Biochemistry and Gene Center, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Lammens
authors.affiliation: Department of Biochemistry and Gene Center, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Stoehr
authors.affiliation: Department of Biochemistry and Gene Center, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Kessler
authors.affiliation: Department of Biochemistry and Gene Center, Ludwig-Maximilians-University, Munich, Germany Center for Integrated Protein Sciences, Munich, Germany hopfner@genzentrum.lmu.de.
authors.forename: Karl-Peter
authors.initials: KP
authors.lastname: Hopfner
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D002597:Chaetomium / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004271:DNA, Fungal / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D000255:Adenosine Triphosphate
keywords: DNA repair; X‐ray crystallography; homologous recombination; microhomology‐mediated end joining; protein DNA complex