Structural mechanism of endonucleolytic processing of blocked DNA ends and hairpins by Mre11-Rad50.

DNA double-strand breaks (DSBs) threaten genome stability and are linked to tumorigenesis in humans. Repair of DSBs requires the removal of attached proteins and hairpins through a poorly understood but physiologically critical endonuclease activity by the Mre11-Rad50 complex. Here, we report cryoelectron microscopy (cryo-EM) structures of the bacterial Mre11-Rad50 homolog SbcCD bound to a protein-blocked DNA end and a DNA hairpin. The structures reveal that Mre11-Rad50 bends internal DNA for endonucleolytic cleavage and show how internal DNA, DNA ends, and hairpins are processed through a similar ATP-regulated conformational state. Furthermore, Mre11-Rad50 is loaded onto blocked DNA ends with Mre11 pointing away from the block, explaining the distinct biochemistries of 3' → 5' exonucleolytic and endonucleolytic incision through the way Mre11-Rad50 interacts with diverse DNA ends. In summary, our results unify Mre11-Rad50's enigmatic nuclease diversity within a single structural framework and reveal how blocked DNA ends and hairpins are processed.

Yeast-based screening of cancer mutations in the DNA damage response protein Mre11 demonstrates importance of conserved capping domain residues.

DNA damage response (DDR) pathways are initiated to prevent mutations from being passed on in the event of DNA damage. Mutations in DDR proteins can contribute to the development and maintenance of cancer cells, but many mutations observed in human tumors have not been functionally characterized. Because a proper response to DNA damage is fundamental to living organisms, DDR proteins and processes are often highly conserved. The goal of this project was to use Saccharomyces cerevisiae as a model for functional screening of human cancer mutations in conserved DDR proteins. After comparing the cancer mutation frequency and conservation of DDR proteins, Mre11 was selected for functional screening. A subset of mutations in conserved residues was analyzed by structural modeling and screened for functional effects in yeast Mre11. Yeast expressing wild type or mutant Mre11 were then assessed for DNA damage sensitivity using hydroxyurea (HU) and methyl methanesulfonate (MMS). The results were further validated in human cancer cells. The N-terminal point mutations tested in yeast Mre11 do not confer sensitivity to DNA damage sensitivity, suggesting that these residues are dispensable for yeast Mre11 function and may have conserved sequence without conserved function. However, a mutation near the capping domain associated with breast and colorectal cancers compromises Mre11 function in both yeast and human cells. These results provide novel insight into the function of this conserved capping domain residue and demonstrate a framework for yeast-based screening of cancer mutations.

Biochemical and structural characterization of analogs of MRE11 breast cancer-associated mutant F237C.

The MRE11-RAD50-NBS1 (MRN) protein complex plays a vital role in DNA double strand break sensing, signaling, and repair. Mutation in any component of this complex may lead to disease as disrupting DNA double strand break repair has the potential to cause translocations and loss of genomic information. Here, we have investigated an MRE11 mutation, F237C, identified in a breast cancer tumor. We found that the analogous mutant of Pyrococcus furiosus Mre11 diminishes both the exonuclease and endonuclease activities of Mre11 in vitro. Solution state NMR experiments show that this mutant causes structural changes in the DNA-bound Mre11 for both exo- and endonuclease substrates and causes the protein to become generally more rigid. Moreover, by comparing the NMR data for this cancer-associated mutant with two previously described Mre11 separation-of-nuclease function mutants, a potential allosteric network was detected within Mre11 that connects the active site to regions responsible for recognizing the DNA ends and for dimerization. Together, our data further highlight the dynamics required for Mre11 nuclease function and illuminate the presence of allostery within the enzyme.

MRN complex is an essential effector of DNA damage repair.

Genome stability can be threatened by both endogenous and exogenous agents. Organisms have evolved numerous mechanisms to repair DNA damage, including homologous recombination (HR) and non-homologous end joining (NHEJ). Among the factors associated with DNA repair, the MRE11-RAD50-NBS1 (MRN) complex (MRE11-RAD50-XRS2 in Saccharomyces cerevisiae) plays important roles not only in DNA damage recognition and signaling but also in subsequent HR or NHEJ repair. Upon detecting DNA damage, the MRN complex activates signaling molecules, such as the protein kinase ataxia-telangiectasia mutated (ATM), to trigger a broad DNA damage response, including cell cycle arrest. The nuclease activity of the MRN complex is responsible for DNA end resection, which guides DNA repair to HR in the presence of sister chromatids. The MRN complex is also involved in NHEJ, and has a species-specific role in hairpin repair. This review focuses on the structure of the MRN complex and its function in DNA damage repair.

Structural insights into DNA double-strand break signaling.

Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of protein-protein signaling interactions that emanate from the DNA damage which are now recognized as a rich basis for anti-cancer therapy development. Deciphering the structures of signaling proteins has been an uphill task owing to their large size and complex domain organization. Recent advances in mammalian protein expression/purification and cryo-EM-based structure determination have led to significant progress in our understanding of these large multidomain proteins. This review is an overview of the structural principles that underlie some of the key signaling proteins that function at the double-strand break site. We also discuss some plausible ideas that could be considered for future structural approaches to visualize and build a more complete understanding of protein dynamics at the break site.

Modes of action of the archaeal Mre11/Rad50 DNA-repair complex revealed by fast-scan atomic force microscopy.

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.

Rad50 zinc hook functions as a constitutive dimerization module interchangeable with SMC hinge.

The human Mre11/Rad50 complex is one of the key factors in genome maintenance pathways. Previous nanoscale imaging by atomic force microscopy (AFM) showed that the ring-like structure of the human Mre11/Rad50 complex transiently opens at the zinc hook of Rad50. However, imaging of the human Mre11/Rad50 complex by high-speed AFM shows that the Rad50 coiled-coil arms are consistently bridged by the dimerized hooks while the Mre11/Rad50 ring opens by disconnecting the head domains; resembling other SMC proteins such as cohesin or condensin. These architectural features are conserved in the yeast and bacterial Mre11/Rad50 complexes. Yeast strains harboring the chimeric Mre11/Rad50 complex containing the SMC hinge of bacterial condensin MukB instead of the RAD50 hook properly functions in DNA repair. We propose that the basic role of the Rad50 hook is similar to that of the SMC hinge, which serves as rather stable dimerization interface.

The MRE11-RAD50-NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair.

Genomic instability in disease and its fidelity in health depend on the DNA damage response (DDR), regulated in part from the complex of meiotic recombination 11 homolog 1 (MRE11), ATP-binding cassette-ATPase (RAD50), and phosphopeptide-binding Nijmegen breakage syndrome protein 1 (NBS1). The MRE11-RAD50-NBS1 (MRN) complex forms a multifunctional DDR machine. Within its network assemblies, MRN is the core conductor for the initial and sustained responses to DNA double-strand breaks, stalled replication forks, dysfunctional telomeres, and viral DNA infection. MRN can interfere with cancer therapy and is an attractive target for precision medicine. Its conformations change the paradigm whereby kinases initiate damage sensing. Delineated results reveal kinase activation, posttranslational targeting, functional scaffolding, conformations storing binding energy and enabling access, interactions with hub proteins such as replication protein A (RPA), and distinct networks at DNA breaks and forks. MRN biochemistry provides prototypic insights into how it initiates, implements, and regulates multifunctional responses to genomic stress.

Interplay with the Mre11-Rad50-Nbs1 complex and phosphorylation by GSK3ß implicate human B-Myb in DNA-damage signaling.

B-Myb, a highly conserved member of the Myb transcription factor family, is expressed ubiquitously in proliferating cells and controls the cell cycle dependent transcription of G2/M-phase genes. Deregulation of B-Myb has been implicated in oncogenesis and loss of genomic stability. We have identified B-Myb as a novel interaction partner of the Mre11-Rad50-Nbs1 (MRN) complex, a key player in the repair of DNA double strand breaks. We show that B-Myb directly interacts with the Nbs1 subunit of the MRN complex and is recruited transiently to DNA-damage sites. In response to DNA-damage B-Myb is phosphorylated by protein kinase GSK3ß and released from the MRN complex. A B-Myb mutant that cannot be phosphorylated by GSK3ß disturbs the regulation of pro-mitotic B-Myb target genes and leads to inappropriate mitotic entry in response to DNA-damage. Overall, our work suggests a novel function of B-Myb in the cellular DNA-damage signalling.