Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex

DNA double-strand breaks (DSBs) trigger activation of the ATM protein kinase, which coordinates cell-cycle arrest, DNA repair and apoptosis. We propose that ATM activation by DSBs occurs in two steps. First, dimeric ATM is recruited to damaged DNA and dissociates into monomers. The Mre11-Rad50-Nbs1 complex (MRN) facilitates this process by tethering DNA, thereby increasing the local concentration of damaged DNA. Notably, increasing the concentration of damaged DNA bypasses the requirement for MRN, and ATM monomers generated in the absence of MRN are not phosphorylated on Ser1981. Second, the ATM-binding domain of Nbs1 is required and sufficient to convert unphosphorylated ATM monomers into enzymatically active monomers in the absence of DNA. This model clarifies the mechanism of ATM activation in normal cells and explains the phenotype of cells from patients with ataxia telangiectasia-like disorder and Nijmegen breakage syndrome.     

Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity

DNA double-strand breaks (DSBs) can be processed by the Mre11-Rad50-Nbs1 (MRN) complex, which is essential to promote ataxia telangiectasia-mutated (ATM) activation. However, the molecular mechanisms linking MRN activity to ATM are not fully understood. Here, using Xenopus laevis egg extract we show that MRN-dependent processing of DSBs leads to the accumulation of short single-stranded DNA oligonucleotides (ssDNA oligos). The MRN complex isolated from the extract containing DSBs is bound to ssDNA oligos and stimulates ATM activity. Elimination of ssDNA oligos results in rapid extinction of ATM activity. Significantly, ssDNA oligos can be isolated from human cells damaged with ionizing radiation and injection of small synthetic ssDNA oligos into undamaged cells also induces ATM activation. These results suggest that MRN-dependent generation of ssDNA oligos, which constitute a unique signal of ongoing DSB repair not encountered in normal DNA metabolism, stimulates ATM activity.     

Adenovirus Regulates Sumoylation of Mre11-Rad50-Nbs1 Components through a Paralog-Specific Mechanism

The Mre11-Rad50-Nbs1 (MRN) complex plays a key role in the DNA damage response, presenting challenges for DNA viruses and retroviruses. To inactivate this complex, adenovirus (Ad) makes use of the E1B-55K and E4-open reading frame 6 (ORF6) proteins for ubiquitin (Ub)-mediated, proteasome-dependent degradation of MRN and the E4-ORF3 protein for relocalization and sequestration of MRN within infected-cell nuclei. Here, we report that Mre11 is modified by the Ub-related modifier SUMO-2 and Nbs1 is modified by both SUMO-1 and SUMO-2. We found that Mre11 and Nbs1 are sumoylated during Ad5 infection and that the E4-ORF3 protein is necessary and sufficient to induce SUMO conjugation. Relocalization of Mre11 and Nbs1 into E4-ORF3 nuclear tracks is required for this modification to occur. E4-ORF3-mediated SUMO-1 conjugation to Nbs1 and SUMO-2 conjugation to Mre11 and Nbs1 are transient during wild-type Ad type 5 (Ad5) infection. In contrast, SUMO-1 conjugation to Nbs1 is stable in cells infected with E1B-55K or E4-ORF6 mutant viruses, suggesting that Ad regulates paralog-specific desumoylation of Nbs1. Inhibition of viral DNA replication blocks deconjugation of SUMO-2 from Mre11 and Nbs1, indicating that a late-phase process is involved in Mre11 and Nbs1 desumoylation. Our results provide direct evidence of Mre11 and Nbs1 sumoylation induced by the Ad5 E4-ORF3 protein and an important example showing that modification of a single substrate by both SUMO-1 and SUMO-2 is regulated through distinct mechanisms. Our findings suggest how E4-ORF3-mediated relocalization of the MRN complex influences the cellular DNA damage response.     

A forward chemical genetic screen reveals an inhibitor of the Mre11-Rad50-Nbs1 complex

The MRN (Mre11-Rad50-Nbs1)-ATM (ataxia-telangiectasia mutated) pathway is essential for sensing and signaling from DNA double-strand breaks. The MRN complex acts as a DNA damage sensor, maintains genome stability during DNA replication, promotes homology-dependent DNA repair and activates ATM. MRN is essential for cell viability, which has limited functional studies of the complex. Small-molecule inhibitors of MRN could circumvent this experimental limitation and could also be used as cellular radio- and chemosensitization compounds. Using cell-free systems that recapitulate faithfully the MRN-ATM signaling pathway, we designed a forward chemical genetic screen to identify inhibitors of the pathway, and we isolated 6-(4-hydroxyphenyl)-2-thioxo-2,3-dihydro-4(1H)-pyrimidinone (mirin, 1) as an inhibitor of MRN. Mirin prevents MRN-dependent activation of ATM without affecting ATM protein kinase activity, and it inhibits Mre11-associated exonuclease activity. Consistent with its ability to target the MRN complex, mirin abolishes the G2/M checkpoint and homology-dependent repair in mammalian cells.     

Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions

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Distinct Roles of the ATR Kinase and the Mre11-Rad50-Nbs1 Complex in the Maintenance of Chromosomal Stability in Arabidopsis

Signaling of chromosomal DNA breaks is of primary importance for initiation of repair and, thus, for global genomic stability. Although the Mre11-Rad50-Nbs1 (MRN) complex is the first sensor of double-strand breaks, its role in double-strand break (DSB) signaling is not fully understood. We report the absence of γ-ray–induced, ATM/ATR-dependent histone H2AX phosphorylation in Arabidopsis thaliana rad50 and mre11 mutants, confirming that the MRN complex is required for H2AX phosphorylation by the ATM and ATR kinases in response to irradiation-induced DSB in Arabidopsis. rad50 and mre11 mutants spontaneously activate a DNA damage response, as shown by the presence of γ-H2AX foci and activation of cell cycle arrest in nonirradiated plants. This response is ATR dependent as shown both by the absence of these spontaneous foci and by the wild-type mitotic indices of double rad50 atr and mre11 atr plants. EdU S-phase labeling and fluorescence in situ hybridization analysis using specific subtelomeric probes point to a replicative S-phase origin of this chromosome damage in the double mutants and not to telomere destabilization. Thus, the data presented here show the exclusive involvement of ATR in DNA damage signaling in MRN mutants and provide evidence for a role for ATR in the avoidance of S-phase DNA damage.     

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.     

Differential regulation of MRN (Mre11-Rad50-Nbs1) complex subunits and telomerase activity in cancer cells

Several lines of evidence suggest that cancer progression is associated with up-regulation or reactivation of telomerase and the underlying mechanism remains an active area of research. The heterotrimeric MRN complex, consisting of Mre11, Rad50 and Nbs1, which is required for the repair of double-strand breaks, plays a key role in telomere length maintenance. In this study, we show significant differences in the levels of expression of MRN complex subunits among various cancer cells and somatic cells. Notably, siRNA-mediated depletion of any of the subunits of MRN complex led to complete ablation of other subunits of the complex. Treatment of leukemia and prostate cancer cells with etoposide lead to increased expression of MRN complex subunits, with concomitant decrease in the levels of telomerase activity, compared to breast cancer cells. These studies raise the possibility of developing anti-cancer drugs targeting MRN complex subunits to sensitize a subset of cancer cells to radio- and/or chemotherapy.     

Relocalization of the Mre11-Rad50-Nbs1 complex by the adenovirus E4 ORF3 protein is required for viral replication

Adenovirus replication is controlled by the relocalization or modification of nuclear protein complexes, including promyelocytic leukemia protein (PML) nuclear domains and the Mre11-Rad50-Nbs1 (MRN) DNA damage machinery. In this study, we demonstrated that the E4 ORF3 protein effects the relocalization of both PML and MRN proteins to similar structures within the nucleus at early times after infection. These proteins colocalize with E4 ORF3. Through the analysis of specific viral mutants, we found a direct correlation between MRN reorganization at early times after infection and the establishment of viral DNA replication domains. Further, the reorganization of MRN components may be uncoupled from the ability of E4 ORF3 to rearrange PML. At later stages of infection, components of the MRN complex disperse within the nucleus, Nbs1 is found within viral replication centers, Rad50 remains localized with E4 ORF3, and Mre11 is degraded. The importance of viral regulation of the MRN complex is underscored by the complementation of E4 mutant viruses in cells that lack Mre11 or Nbs1 activity. These results illustrate the importance of nuclear organization in virus growth and suggest that E4 ORF3 regulates activities in both PML nuclear bodies and the MRN complex to stimulate the viral replication program.     

The fission yeast Rad32 (Mre11)-Rad50-Nbs1 complex is required for the S-phase DNA damage checkpoint

Mre11, Rad50, and Nbs1 form a conserved heterotrimeric complex that is involved in recombination and DNA damage checkpoints. Mutations in this complex disrupt the S-phase DNA damage checkpoint, the checkpoint which slows replication in response to DNA damage, and cause chromosome instability and cancer in humans. However, how these proteins function and specifically where they act in the checkpoint signaling pathway remain crucial questions. We identified fission yeast Nbs1 by using a comparative genomic approach and showed that the genes for human Nbs1 and fission yeast Nbs1 and that for their budding yeast counterpart, Xrs2, are members of an evolutionarily related but rapidly diverging gene family. Fission yeast Nbs1, Rad32 (the homolog of Mre11), and Rad50 are involved in DNA damage repair, telomere regulation, and the S-phase DNA damage checkpoint. However, they are not required for G(2) DNA damage checkpoint. Our results suggest that a complex of Rad32, Rad50, and Nbs1 acts specifically in the S-phase branch of the DNA damage checkpoint and is not involved in general DNA damage recognition or signaling.     

Ataxia telangiectasia-mutated damage-signaling kinase- and proteasome-dependent destruction of Mre11-Rad50-Nbs1 subunits in Simian virus 40-infected primate cells

Although the mechanism of simian virus 40 (SV40) DNA replication has been extensively investigated with cell extracts, viral DNA replication in productively infected cells utilizes additional viral and host functions whose interplay remains poorly understood. We show here that in SV40-infected primate cells, the activated ataxia telangiectasia-mutated (ATM) damage-signaling kinase, gamma-H2AX, and Mre11-Rad50-Nbs1 (MRN) assemble with T antigen and other viral DNA replication proteins in large nuclear foci. During infection, steady-state levels of MRN subunits decline, although the corresponding mRNA levels remain unchanged. A proteasome inhibitor stabilizes the MRN complex, suggesting that MRN may undergo proteasome-dependent degradation. Analysis of mutant T antigens with disrupted binding to the ubiquitin ligase CUL7 revealed that MRN subunits are stable in cells infected with mutant virus or transfected with mutant viral DNA, implicating CUL7 association with T antigen in MRN proteolysis. The mutant genomes produce fewer virus progeny than the wild type, suggesting that T antigen-CUL7-directed proteolysis facilitates virus propagation. Use of a specific ATM kinase inhibitor showed that ATM kinase signaling is a prerequisite for proteasome-dependent degradation of MRN subunits as well as for the localization of T antigen and damage-signaling proteins to viral replication foci and optimal viral DNA replication. Taken together, the results indicate that SV40 infection manipulates host DNA damage-signaling to reprogram the cell for viral replication, perhaps through mechanisms related to host recovery from DNA damage.     

Mre11-Rad50 complex crystals suggest molecular calisthenics

Recently published crystal structures of different Mre11 and Rad50 complexes show the arrangement of these proteins and imply dramatic ligand-induced rearrangements with important functional consequences.     

The Mre11-Rad50-Xrs2 Complex Is Required for Yeast DNA Postreplication Repair

Yeast DNA postreplication repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 in vivo. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass.     

Mre11-Rad50-Nbs1 conformations and the control of sensing, signaling, and effector responses at DNA double-strand breaks

Repair and integrity of DNA ends at breaks, replication forks and telomeres are essential for life; yet, paradoxically, these responses are, in many cases, controlled by a single protein complex, Mre11-Rad50-Nbs1 (MRN). The MRN complex consists of dimers of each subunit and this heterohexamer controls key sensing, signaling, regulation, and effector responses to DNA double-strand breaks including ATM activation, homologous recombinational repair, microhomology-mediated end joining and, in some organisms, non-homologous end joining. We propose that this is possible because each MRN subunit can exist in three or more distinct states; thus, the trimer of MRN dimers can exist in a stunning 6(3) or 216 states, a number that can be expanded further when post-translational modifications are taken into account. MRN can therefore be considered as a molecular computer that effectively assesses optimal responses and pathway choice based upon its states as set by cell status and the nature of the DNA damage. This extreme multi-state concept demands a paradigm shift from striving to understand DNA damage responses in separate terms of signaling, checkpoint, and effector proteins: we must now endeavor to characterize conformational and assembly states of MRN and other DNA repair machines that couple, coordinate, and control biological outcomes. Addressing the emerging challenge of gaining a detailed molecular understanding of MRN and other multi-state dynamic DNA repair machines promises to provide opportunities to develop master keys for controlling cell biology with probable impacts on therapeutic interventions.     

Biochemical characterization of bacteriophage T4 Mre11-Rad50 complex

The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50- and Mn(2+)-dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence of Mre11 and dsDNA increases ATP hydrolysis by 20-fold. The ATP hydrolysis reaction exhibits positive cooperativity with Hill coefficients ranging from 1.4 for Rad50 alone to 2.4 for the Rad50-Mre11-DNA complex. Kinetic assays suggest that approximately four nucleotides are removed per ATP hydrolyzed. Directionality assays indicate that the prevailing activity is a 3' to 5' dsDNA exonuclease, which is incompatible with the proposed role of MR in the production of 3' ssDNA ends. Interestingly, we found that in the presence of a recombination mediator protein (UvsY) and ssDNA-binding protein (gp32), Mre11 is capable of using Mg(2+) as a cofactor for its nuclease activity. Additionally, the Mg(2+)-dependent nuclease activity, activated by UvsY and gp32, results in the formation of endonuclease reaction products. These results suggest that gp32 and UvsY may alter divalent cation preference and facilitate the formation of a 3' ssDNA overhang, which is a necessary intermediate for recombination-mediated double-strand break repair.     

Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template.

The Mre11-Rad50-Nbs1 (MRN) complex is providing paradigm-shifting results of exceptional biomedical interest. MRN is among the earliest respondents to DNA double-strand breaks (DSBs), and MRN mutations cause the human cancer predisposition diseases Nijmegen breakage syndrome and ataxia telangiectasia-like disorder (ATLD). MRN's 3-protein multidomain composition promotes its central architectural, structural, enzymatic, sensing, and signaling functions in DSB responses. To organize the MRN complex, the Mre11 exonuclease directly binds Nbs1, DNA, and Rad50. Rad50, a structural maintenance of chromosome (SMC) related protein, employs its ATP-binding cassette (ABC) ATPase, Zn hook, and coiled coils to bridge DSBs and facilitate DNA end processing by Mre11. Contributing to MRN regulatory roles, Nbs1 harbors N-terminal phosphopeptide interacting FHA and BRCT domains, as well as C-terminal ataxia telangiectasia mutated (ATM) kinase and Mre11 interaction domains. Current emerging structural and biological evidence suggests that MRN has 3 coupled critical roles in DSB sensing, stabilization, signaling, and effector scaffolding: (1) expeditious establishment of protein--nucleic acid tethering scaffolds for the recognition and stabilization of DSBs; (2) initiation of DSB sensing, cell-cycle checkpoint signaling cascades, and establishment of epigenetic marks via the ATM kinase; and (3) functional regulation of chromatin remodeling in the vicinity of a DSB.     

The Mre11-Rad50-Xrs2 Protein Complex Facilitates Homologous Recombination-Based Double-Strand Break Repair in Saccharomyces cerevisiae

Saccharomyces cerevisiae mre11Delta mutants are profoundly deficient in double-strand break (DSB) repair, indicating that the Mre11-Rad50-Xrs2 protein complex plays a central role in the cellular response to DNA DSBs. In this study, we examined the role of the complex in homologous recombination, the primary mode of DSB repair in yeast. We measured survival in synchronous cultures following irradiation and scored sister chromatid and interhomologue recombination genetically. mre11Delta strains were profoundly sensitive to ionizing radiation (IR) throughout the cell cycle. Mutant strains exhibited decreased frequencies of IR-induced sister chromatid and interhomologue recombination, indicating a general deficiency in homologous recombination-based DSB repair. Since a nuclease-deficient mre11 mutant was not impaired in these assays, it appears that the role of the S. cerevisiae Mre11-Rad50-Xrs2 protein complex in facilitating homologous recombination is independent of its nuclease activities.     

Stable maintenance of the Mre11-Rad50-Nbs1 complex is sufficient to restore the DNA double-strand break response in cells lacking RecQL4 helicase activity

The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund–Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.