MRN domains, complex assembly, and mutations. (a) Key domains of MRN subunits MRE11 (orange), RAD50 ( green), and NBS1 (cyan). Mutated sites (red arrows) for human inherited diseases (color coded for ATLD, NBS, and NBSLD; mutations corresponding to each disease are color coded with respective abbreviations) are shown on each subunit with respective substitutions, where X represents the stop codon. (b) Modeled assembly of MRN complex and dsDNA based on experimentally derived partial crystal structures (PDB IDs: 5F3W, 5GOX, and 3HUE; color coding as in panel a) showing a dimer of a heterotrimer. The allosterically linked Zn-hook is highlighted in the inset along with CxxC motifs (red ) on two RAD50 monomers. (c) The extent of mutations (reported as mutation factor) in subunits of the MRN complex in cancers documented in the International Cancer Genome Consortium (ICGC) projects. A mutation factor is defined as a number of observed mutations multiplied by the fraction of mutation-positive cases. ICGC projects with high mutation factors (>15 in total) include the following: #13, Breast Cancer, ER+ and HER2−, European Union/United Kingdom; #20, Esophageal Adenocarcinoma, United Kingdom; #28 Liver Cancer, China; #31, Liver Cancer, RIKEN, Japan; #36, Malignant Lymphoma, Germany; #52, Skin Adenocarcinoma, Brazil; #53, Skin Cancer, Australia; and #55, Soft Tissue Cancer, Leiomyosarcoma, France. A complete list of the 58 projects is provided in Supplemental Table 1. Abbreviations: ABM, ATM binding motif; ATLD, ataxia-telangiectasia-like disorder; BRCT, BRCA1 C-terminal domain; dsDNA, double-stranded DNA; FHA, forkhead-associated domain; MBM, MRE11 binding motif; MRE11, meiotic recombination 11 homolog 1; NBS, Nijmegen breakage syndrome; NBSLD, NBS-like disorder; PDB, Protein Data Bank; RAD50, DNA repair protein RAD50; RBD, RAD50 binding domain.

The Mre11–Rad50 (MR) heterotetramer: DNA-binding interface, ATP-mediated conformational changes, and a unified model for MRN’s functions in DSB resection (color coding as in ) are shown. (a) Crystal structures of ATPγS-bound Methanococcus jannaschii (Mj) Mre11—Rad50–DNA complex (PDB ID: 5F3W) and the symmetric (PDB ID: 3DSC) and asymmetric (PDB ID: 3DSD) Pyrococcus furiosus Mre11 DNA-bound states, highlighting the DNA-binding interface at the Rad50 and Mre11 dimerization interface. (b) Crystal structures of the MR complex in the nucleotide-free state (PDB ID: 3QG5, Thermotoga maritima MR) and in the ATPγS-bound state (PDB ID: 3AV0, MjMR), highlighting the conformational switch in the MRN core complex. (c) Human CtIP model from the recent crystal structure of CtIP N-terminal tetramerization domain (PDB ID: 4D2H). Plausible NBS1 binding sites on four CtIP and C-terminal DBMs, along with CxxC motifs and Zn coordination, are shown. (d ) A testable pathway for DSB resection by the MRN complex. For clarity, only MRE11 (M) and RAD50 (R) subunits are shown. First, two molecules of MRN bind to DSB ends in an ATP-bound state, with MRE11 nuclease activity inhibited by the closed RAD50 conformation. Then, MRN forms an interlinked complex through a RAD50 coil swap. At this point, CtIP–NBS1 interaction allows MRN dimers to slide away from the break, and MRE11 makes the initial licensing cut through its endonuclease activity. Upon complete ATP hydrolysis, the RAD50 dimerization interface opens and dsDNA becomes accessible to the MRE11 exonuclease. DNA resection begins in the 3΄–5΄ direction and proceeds toward the break. Upon reaching the break, the MRN complex may be dislodged from DSB with the help of CtIP and NBS1 (NBS1 not shown for clarity). Because only M and R are shown in the model, NBS1 (N) is colored gray in MRN notation. Abbreviations: CTD, C-terminal domain; CtIP, C-terminal binding protein–interacting protein; DBMs, DNA binding motifs; DSBs, double-strand breaks; dsDNA, double-stranded DNA; MRE11, meiotic recombination 11 homolog 1; MRN, MRE11–RAD50–NBS1 complex; NBS1, Nijmegen breakage syndrome protein 1; NTD, N-terminal domain; PDB, Protein Data Bank; RAD50, DNA repair protein RAD50.

The MRN complex activates possible (alternative) pathways at DSBs. The MRN complex can initially be recruited to a DSB by RAD17 and activates the ATM kinase. Once activated, ATM phosphorylates downstream substrates including MDC1, which in turn recruits MRN, and the ATM signal is further amplified at the DSB. ATM checkpoint signaling leads to cell cycle regulation through CHK2 activation ( far left). On the basis of the cell cycle phase and nature of the DSB ends, DSBs are channeled through either HR or NHEJ for repair (central paths). In HR, the MRN complex initiates the 5΄ resection followed by long-range resection by EXO1 or DNA2. The resulting 3΄ ssDNA overhangs are loaded with ssDNA-binding protein RPA. The RPA-coated ssDNA can activate a second checkpoint pathway: ATR signaling. ATR is recruited to the RPA-coated ssDNA through ATRIP and RPA interaction. Then the RAD17–RFC clamp loading complex loads the 9–1–1 (RAD9–HUS1–RAD1) checkpoint clamp at the ssDNA and dsDNA junction on the RPA-coated ssDNA arms. The ATR activator TopBP1 is loaded by RAD9, leading to activation of CHK1 kinase and cell cycle regulation. For high-fidelity DSB repair, the BRCA2–RAD51 complex nucleates the formation of RAD51 nucleofilaments and displaces RPA from ssDNA. RAD51 nucleofilaments promote homology search and strand invasion in association with BRCA1–BARD1 for error-free HR repair through the resolution of repair intermediates. The two NHEJ pathways (right) depend upon DNA-PKcs and MRN–CtIP. In C-NHEJ, DSB ends are bluntly fused through Ku70/80–DNA-PKcs complex tethering, Artemis-mediated end processing, ligase IV, and XLF-XRCC4 scaffolding factor–mediated ligation. The C-NHEJ is independent of the MRN complex; however, MMEJ operates through MRN–CtIP-mediated resection followed by priming based on microhomology, flap removal, and gap filling. Abbreviations: ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiectasia and RAD3-related; ATRIP, ATR-interacting protein; C-NHEJ, canonical nonhomologous end joining; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; DSB, double-strand break; HR, homologous recombination; MMEJ, microhomology-mediated end joining; MRE11, meiotic recombination 11 homolog 1; MRN, MRE11–RAD50–NBS1 complex; NBS1, Nijmegen breakage syndrome protein 1; NHEJ, nonhomologous end joining; RAD50, DNA repair protein RAD50; RFC, replication factor C; RPA, replication protein A; ssDNA, single-stranded DNA.

The MRN complex with RPA and ATR orchestrates replication fork protection, remodeling, and restart as well as HR rescue of collapsed forks. At stalled forks, the MRN complex participates in fork protection through non-nuclease functions (e.g., DNA tethering), and its ssDNA endonuclease activity is inhibited by RPA. RPA-coated ssDNA initiates ATR checkpoint signaling that provides cells time for repair and restart. Replication barriers are removed by remodeling through fork reversal that must then be processed for fork restart. The nascent ssDNA at reversed forks is protected against MRN exonuclease activity by RAD51 loading in association with BRCA2. For restart, MRE11 excision of one strand may facilitate fork reversal and restart. Unrepaired collapsed forks can become DSBs where the MRN complex initiates homologous recombination by initiating resection. Abbreviations: ATR, ataxia-telangiectasia and RAD3-related; ATRIP, ATR-interacting protein; MRE11, meiotic recombination 11 homolog 1; MRN, MRE11–RAD50–NBS1 complex; NBS1, Nijmegen breakage syndrome protein 1; RAD50, DNA repair protein RAD50; RPA, replication protein A; ssDNA, single-stranded DNA.

Telomere structure and dynamics regulate MRN interactions and distinguish chromosome ends from breaks. Human telomeres consist of a long stretch of repetitive TTAGGG sequences and interacting proteins. Telomeres forming from the replication of a leading strand result in tails with blunt ends, whereas the replication of a lagging strand generates telomeres with 3΄ overhangs. The 5΄ resection of a blunt-ended telomere inhibits telomere fusion through NHEJ. The telomeres with 3΄ overhangs form transient T-loop structures. The shelterin complex, which helps protect telomere ends, consists of telomeric repeat-binding factors 1 and 2 (TRF1 and TRF2), repressor and activator protein (RAP1), TRF1-interacting nuclear protein 2 (TIN2), protection of telomeres 1 (POT1), and TIN2- and POT1-interacting protein (TPP1). Abbreviations: ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiectasia and RAD3-related; CtIP, C-terminal binding protein–interacting protein; MRE11, meiotic recombination 11 homolog 1; MRN, MRE11–RAD50–NBS1 complex; NBS1, Nijmegen breakage syndrome protein 1; NHEJ, nonhomologous end joining; RAD50, DNA repair protein RAD50; T-loop, telomere loop.

MRN in host and virus responses. (a) The MRN complex inhibits viral replication via pathways linking DNA repair proteins and the innate immune response. The interaction between the MRN complex and viral DNA leads to local ATM activation that avoids cell cycle arrest and apoptosis, which is characteristic of global ATM signaling in DNA damage. The MRN complex promotes the formation of viral DNA concatemers (randomly fused long chains of viral DNA) in association with NHEJ proteins to help inhibit viral replication. MRN components furthermore promote the innate immune response by inducing interferon and cytokine production through pathways involving STING, IRF3, and NF-κβ. (b) Viruses neutralize the MRN complex by relocalization and/or degradation. The viral protein complex of E4-ORF6 and E1B-55K directs the MRN complex for degradation through ubiquitination (Ub). The viral protein E4-ORF3 relocalizes the MRN complex to the nucleus through sumoylation (Su) to promote viral replication by limiting MRN-promoted viral DNA concatemer formation. Abbreviations: ATM, ataxia-telangiectasia mutated; CARD9, caspase recruitment domain-containing protein 9; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; E1B-55K, a 55-kDa protein of adenovirus E1B gene; E4-ORF3, early region 4 of adenovirus open reading frame 3 protein; E4-ORF6, early region 4 of adenovirus open reading frame 6 protein; IFNs, type I interferons; IL-1β, interleukin 1 beta; IL-33, interleukin 33; IRF3, interferon regulatory factor 3; MRE11, meiotic recombination 11 homolog 1; MRN, MRE11–RAD50–NBS1 complex; NBS1, Nijmegen breakage syndrome protein 1; NF-κβ, nuclear factor κβ; NHEJ, nonhomologous end joining; RAD50, DNA repair protein RAD50; STING, stimulator of interferon genes.