Crystal structure of the Mre11–Rad50–ATPγS complex: understanding the interplay between Mre11 and Rad50

Abstract

Communication between Mre11 and Rad50 in the MR complex is critical for the sensing, damage signaling, and repair of DNA double-strand breaks. To understand the basis for interregulation between Mre11 and Rad50, we determined the crystal structure of the Mre11-Rad50-ATPgS complex. Mre11 brings the two Rad50 molecules into close proximity and promotes ATPase activity by (1) holding the coiled-coil arm of Rad50 through its C-terminal domain, (2) stabilizing the signature motif and P loop of Rad50 via its capping domain, and (3) forming a dimer through the nuclease domain. ATP-bound Rad50 negatively regulates the nuclease activity of Mre11 by blocking the active site of Mre11. Hydrolysis of ATP disengages Rad50 molecules, and, concomitantly, the flexible linker that connects the C-terminal domain and the capping domain of Mre11 undergoes substantial conformational change to relocate Rad50 and unmask the active site of Mre11. Our structural and biochemical data provide insights into understanding the interplay between Mre11 and Rad50 to facilitate efficient DNA damage repair.

Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins

“…Also, Rad50 negatively regulates Mre11 nucleolytic activity, and repression was alleviated upon ATP hydrolysis by Rad50, indicating that ATP and ADP function as allosteric effectors (46,47). In agreement with these findings, structural studies show that Rad50 negatively regulates Mre11 nuclease activity by blocking the active site of Mre11, and hydrolysis of ATP disengages Rad50 subunit, thus unmasking the active site of Mre11 (48). Furthermore, the human Mre11-Rad50-Nbs1 complex has been shown to possess weak ATP-stimulated DNA unwinding and does not need ends for DNA binding activity (49,50), and hMre11 by itself can bind both ssDNA and dsDNA and catalyze the annealing of complementary ssDNA molecules (51).…”

“…Stimulation of ScMre11 Catalyzed DNA Unwinding by Rad50, Xrs2, and Sae2 Proteins-We showed previously that the ScMre11 nuclease activity is allosterically regulated by Rad50 and ATP (46), which has been corroborated by x-ray crystallographic studies (48). These observations prompted us to investigate whether the DNA unwinding activity of ScMre11 is also subjected to regulation by proteins that are known to interact with Mre11 during DSB end repair.…”

The Rad50 hook domain regulates DNA damage signaling and tumorigenesis

“…In cis, the globular domain could bridge two DNA ends in a manner that would promote NHEJ (Williams et al 2008). In this mode, end-to-end DNA bridging is effected by adjacent monomeric components of the dimeric globular domain (Lammens et al 2011;Lim et al 2011).…”

Structure of the catalytic domain of Mre11 fromChaetomium thermophilum

“…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.…”