Structure of Mre11–Nbs1 complex yields insights into ataxia-telangiectasia–like disease mutations and DNA damage signaling

Abstract

SummaryThe Mre11–Rad50–Nbs1 (MRN) complex tethers, processes and signals DNA double strand breaks, promoting genomic stability. To understand the functional architecture of MRN, we determined the crystal structures of the Schizosaccharomyces pombe Mre11 dimeric catalytic domain alone and in complex with a fragment of Nbs1. Two Nbs1 subunits stretch around the outside of Mre11’s nuclease domains, with one subunit additionally bridging and locking the Mre11 dimer via a highly conserved asymmetrical binding motif. Our results reveal that Mre11 forms a flexible dimer and suggest that Nbs1 is not only a checkpoint adaptor, but also functionally impacts on Mre11-Rad50. Clinical mutations in Mre11 are located along the Nbs1 interaction sites and weaken the Mre11–Nbs1 interaction. However, they differentially affect DNA repair and telomere maintenance in Saccharomyces cerevisiae, potentially providing insight into their different human disease pathologies.

Structure of the catalytic domain of Mre11 fromChaetomium thermophilum

“…We crystallized the catalytic domain of CtMre11 (CtMre11 CD ; amino acids 4-412) and determined the structure by molecular replacement using S. pombe Mre11 (SpMre11 CD ) as the search model (PDB entry 4fbq; Schiller et al, 2012). The crystallization screen contained the MRN head complex (MRN HC ) and, presumably owing to proteolysis, CtMre11 CD crystals formed.…”

“…Although comparison of these structures reveals a highly conserved overall shape of the protein, consisting of an N-terminal phosphodiesterase domain followed by a capping domain, the dimer angle between the Mre11 protomers can adopt remarkably different conformations (Schiller et al, 2014). In eukaryotes, the dimer angle is stabilized by latching loops that provide a critical interaction site for Nbs1 with Mre11 (Schiller et al, 2012;Park et al, 2011). However, a substantial portion of the functionally important, eukaryote-specific latching loops remained disordered in previously determined structures (Schiller et al, 2012;Park et al, 2011).…”

“…In eukaryotes, the dimer angle is stabilized by latching loops that provide a critical interaction site for Nbs1 with Mre11 (Schiller et al, 2012;Park et al, 2011). However, a substantial portion of the functionally important, eukaryote-specific latching loops remained disordered in previously determined structures (Schiller et al, 2012;Park et al, 2011). Here, we present the crystal structure of the Mre11 catalytic domain dimer from the thermophilic eukaryote Chaetomium thermophilum (CtMre11 CD ) at 2.8 Å resolution.…”

“…The structure of the C. thermophilum Mre11 catalytic domain (CtMre11 CD ; amino acids 4-412) was solved by molecular replacement with Phaser (McCoy et al, 2007). The search model was the structure of monomeric Schizosaccharomyces pombe Mre11 (PDB entry 4fbq; Schiller et al, 2012), which was co-crystallized with an Nbs1 construct. The structure was refined with PHENIX, accounting for twinning (Adams et al, 2010), in combination with manual model building using Coot (Emsley & Cowtan, 2004;Emsley et al, 2010).…”

“…In eukaryotes, the Mre11-Rad50-Nbs1 (MRN) complex plays a key role in the early steps of DSB repair, and its function in the initial detection and processing of DNA ends is important for the choice between resection-dependent (HR, MMEJ) and resection-independent (NHEJ) pathways (Lisby et al, 2004;Truong et al, 2013;Chiruvella et al, 2013;Shibata et al, 2014). MRN consists of a dimer of Mre11, two Rad50s and, in eukaryotes, Nbs1 (Lammens et al, 2011;Schiller et al, 2012;Mö ckel et al, 2012;Lim et al, 2011;Arthur et al, 2004;Das et al, 2010;Limbo et al, 2012). The Mre11 nuclease forms the enzymatically active centre of the complex.…”