The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability

The Mre11/Rad50/Nbs (MRN) complex and the two protein kinases ATM and ATR play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. It has been previously shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. Here, we have analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations.     

Competition between the Rad50 complex and the Ku heterodimer reveals a role for Exo1 in processing double-strand breaks but not telomeres

The Mre11-Rad50-Nbs1(Xrs2) complex and the Ku70-Ku80 heterodimer are thought to compete with each other for binding to DNA ends. To investigate the mechanism underlying this competition, we analyzed both DNA damage sensitivity and telomere overhangs in Schizosaccharomyces pombe rad50-d, rad50-d pku70-d, rad50-d exo1-d, and pku70-d rad50-d exo1-d cells. We found that rad50 exo1 double mutants are more methyl methanesulfonate (MMS) sensitive than the respective single mutants. The MMS sensitivity of rad50-d cells was suppressed by concomitant deletion of pku70+. However, the MMS sensitivity of the rad50 exo1 double mutant was not suppressed by the deletion of pku70+. The G-rich overhang at telomere ends in taz1-d cells disappeared upon deletion of rad50+, but the overhang reappeared following concomitant deletion of pku70+. Our data suggest that the Rad50 complex can process DSB ends and telomere ends in the presence of the Ku heterodimer. However, the Ku heterodimer inhibits processing of DSB ends and telomere ends by alternative nucleases in the absence of the Rad50-Rad32 protein complex. While we have identified Exo1 as the alternative nuclease targeting DNA break sites, the identity of the nuclease acting on the telomere ends remains elusive.     

Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11

Mre11-Rad50-Nbs1 (MRN) complex involvement in nonhomologous end joining (NHEJ) is controversial. The MRN complex is required for NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe. In vertebrates, Mre11, Rad50, and Nbs1 are essential genes, and studies have been limited to cells carrying hypomorphic mutations in Mre11 or Nbs1, which still perform several MRN complex-associated activities. In this study, we analyze the effects of Mre11 loss on the mechanism of vertebrate NHEJ by using a chromatinized plasmid double-strand break (DSB) repair assay in cell-free extracts from Xenopus laevis. Mre11-depleted extracts are able to support efficient NHEJ repair of DSBs regardless of the end structure. Mre11 depletion does not alter the kinetics of end joining or the type and frequency of junctions found in repaired products. Finally, Ku70-independent end-joining events are not affected by Mre11 loss. Our data demonstrate that the MRN complex is not required for efficient and accurate NHEJ-mediated repair of DSBs in this vertebrate system.     

The Drosophila Mre11/Rad50 complex is required to prevent both telomeric fusion and chromosome breakage

The MRN complex consists of the two evolutionarily conserved components Mre11 and Rad50 and the third less-conserved component Nbs1/Xrs2. This complex mediates telomere maintenance in addition to a variety of functions in response to DNA double-strand breaks, including homologous recombination, nonhomologous end joining (NHEJ), and activation of DNA damage checkpoints. Mutations in the Mre11 gene cause the human ataxia-telangiectasia-like disorder (ATDL). Here, we show that null mutations in the Drosophila mre11 and rad50 genes cause both telomeric fusion and chromosome breakage. Moreover, we demonstrate that these mutations are in the same epistasis group required for telomere capping and mitotic chromosome integrity. Using an antibody against Rad50, we show that this protein is uniformly distributed along mitotic chromosomes, and that Rad50 is unstable in the absence of its binding partner Mre11. To define the roles of rad50 and mre11 in telomere protection, mutant chromosome preparations were immunostained for both HP1 and HOAP, two proteins that protect Drosophila telomeres from fusion. Cytological analysis revealed that mutations in rad50 and mre11 drastically reduce accumulation of HOAP and HP1 at telomeres. This suggests that the MRN complex protects Drosophila telomeres by facilitating recruitment of HOAP and HP1 at chromosome ends.     

The role of Schizosaccharomyces pombe Rad32, the Mre11 homologue, and other DNA damage response proteins in non-homologous end joining and telomere length maintenance.

The Schizosaccharomyces pombe homologue of Mre11, Rad32, is required for repair of UV- and ionising radiation-induced DNA damage and meiotic recombination. In this study we have investigated the role of Rad32 and other DNA damage response proteins in non-homologous end joining (NHEJ) and telomere length maintenance in S.pombe. We show that NHEJ in S.pombe occurs by an error-prone mechanism, in contrast to the accurate repair observed in Saccharomyces cerevisiae. Deletion of the rad32 gene results in a modest reduction in NHEJ activity and the remaining repair events that occur are accurate. Mutations in two of the phosphoesterase motifs in Rad32 have no effect on the efficiency or accuracy of end joining, suggesting that the role of Rad32 protein may be to recruit another nuclease(s) for processing during the end joining reaction. We also analysed NHEJ in other DNA damage response mutants and showed that the checkpoint mutant rad3-d and two recombination mutants defective in rhp51 and rhp54 (homologues of S.cerevisiae RAD51 and RAD54, respectively) are not affected. However disruption of rad22, rqh1 and rhp9 / crb2 (homologues of the S.cerevisiae RAD52, SGS1 and RAD9 genes) resulted in increased NHEJ activity. Telomere lengths in the rad32, rhp9 and rqh1 null alleles were reduced to varying extents intermediate between the lengths observed in wild-type and rad3 null cells.     

Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair.

To study the role of Rad50 in the DNA damage response, we cloned and deleted the Schizosaccharomyces pombe RAD50 homologue. The deletion is sensitive to a range of DNA-damaging agents and shows dynamic epistatic interactions with other recombination-repair genes. We show that Rad50 is necessary for recombinational repair of the DNA lesion at the mating-type locus and that rad50Delta shows slow DNA replication. We also find that Rad50 is not required for slowing down S phase in response to hydroxy urea or methyl methanesulfonate (MMS) treatment. Interestingly, in rad50Delta cells, the recombination frequency between two homologous chromosomes is increased at the expense of sister chromatid recombination. We propose that Rad50, an SMC-like protein, promotes the use of the sister chromatid as the template for homologous recombinational repair. In support of this, we found that Rad50 functions in the same pathway for the repair of MMS-induced damage as Rad21, the homologue of the Saccharomyces cerevisiae Scc1 cohesin protein. We speculate that Rad50 interacts with the cohesin complex during S phase to assist repair and possibly re-initiation of replication after replication fork collapse.     

A novel recombination pathway initiated by the Mre11/Rad50/Nbs1 complex eliminates palindromes during meiosis in Schizosaccharomyces pombe

DNA palindromes are rare in humans but are associated with meiosis-specific translocations. The conserved Mre11/Rad50/Nbs1 (MRN) complex is likely directly involved in processing palindromes through the homologous recombination pathway of DNA repair. Using the fission yeast Schizosaccharomyces pombe as a model system, we show that a 160-bp palindrome (M-pal) is a meiotic recombination hotspot and is preferentially eliminated by gene conversion. Importantly, this hotspot depends on the MRN complex for full activity and reveals a new pathway for generating meiotic DNA double-strand breaks (DSBs), separately from the Rec12 (ortholog of Spo11) pathway. We show that MRN-dependent DSBs are formed at or near the M-pal in vivo, and in contrast to the Rec12-dependent breaks, they appear early, during premeiotic replication. Analysis of mrn mutants indicates that the early DSBs are generated by the MRN nuclease activity, demonstrating the previously hypothesized MRN-dependent breakage of hairpins during replication. Our studies provide a genetic and physical basis for frequent translocations between palindromes in human meiosis and identify a conserved meiotic process that constantly selects against palindromes in eukaryotic genomes.     

The Role of MRN in the S-Phase DNA Damage Checkpoint Is Independent of Its Ctp1-dependent Roles in Double-Strand Break Repair and Checkpoint Signaling

The Mre11-Rad50-Nbs1 (MRN) complex has many biological functions: processing of double-strand breaks in meiosis, homologous recombination, telomere maintenance, S-phase checkpoint, and genome stability during replication. In the S-phase DNA damage checkpoint, MRN acts both in activation of checkpoint signaling and downstream of the checkpoint kinases to slow DNA replication. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5′ resection to create single-stranded DNA that is required for both signaling and homologous recombination. However, it is unclear whether resection is essential for all of the cellular functions of MRN. To dissect the various roles of MRN, we performed a structure–function analysis of nuclease dead alleles and potential separation-of-function alleles analogous to those found in the human disease ataxia telangiectasia-like disorder, which is caused by mutations in Mre11. We find that several alleles of rad32 (the fission yeast homologue of mre11), along with ctp1Δ, are defective in double-strand break repair and most other functions of the complex, but they maintain an intact S phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads us to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function.     

Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks

The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.     

Phosphorylation-regulated binding of Ctp1 to Nbs1 is critical for repair of DNA double-strand breaks

Repair of DNA double-strand breaks (DSBs) is critical for cell survival and for maintaining genome stability in eukaryotes. In Schizosaccharomyces pombe, the Mre11-Rad50-Nbs1 (MRN) complex and Ctp1 cooperate to perform the initial steps that process and repair these DNA lesions via homologous recombination (HR). While Ctp1 is recruited to DSBs in an MRN-dependent manner, the specific mechanism of this process remained unclear. We recently found that Ctp1 is phosphorylated on a domain rich in putative Casein kinase 2 (CK2) phosphoacceptor sites that resembles the SDTD repeats of Mdc1. Furthermore, phosphorylation of this motif is required for interaction with the FHA domain of Nbs1 that localizes Ctp1 to DSB sites. Here, we review and discuss these findings, and we present new data that further characterize the cellular consequences of mutating CK2 phosphorylation motifs of Ctp1, including data showing that these sites are critical for meiosis.     

A recombination repair gene of Schizosaccharomyces pombe, rhp57, is a functional homolog of the Saccharomyces cerevisiae RAD57 gene and is phylogenetically related to the human XRCC3 gene

To identify Schizosaccharomyces pombe genes involved in recombination repair, we identified seven mutants that were hypersensitive to both methyl methanesulfonate (MMS) and gamma-rays and that contained mutations that caused synthetic lethality when combined with a rad2 mutation. One of the mutants was used to clone the corresponding gene from a genomic library by complementation of the MMS-sensitive phenotype. The gene obtained encodes a protein of 354 amino acids whose sequence is 32% identical to that of the Rad57 protein of Saccharomyces cerevisiae. An rhp57 (RAD57 homolog of S. pombe) deletion strain was more sensitive to MMS, UV, and gamma-rays than the wild-type strain and showed a reduction in the frequency of mitotic homologous recombination. The MMS sensitivity was more severe at lower temperature and was suppressed by the presence of a multicopy plasmid bearing the rhp51 gene. An rhp51 rhp57 double mutant was as sensitive to UV and gamma-rays as an rhp51 single mutant, indicating that rhp51 function is epistatic to that of rhp57. These characteristics of the rhp57 mutants are very similar to those of S. cerevisiae rad57 mutants. Phylogenetic analysis suggests that Rhp57 and Rad57 are evolutionarily closest to human Xrcc3 of the RecA/Rad51 family of proteins.     

A 160-bp palindrome is a Rad50.Rad32-dependent mitotic recombination hotspot in Schizosaccharomyces pombe

Palindromic sequences can form hairpin and cruciform structures that pose a threat to genome integrity. We found that a 160-bp palindrome (an inverted repeat of 80 bp) conferred a mitotic recombination hotspot relative to a control nonpalindromic sequence when inserted into the ade6 gene of Schizosaccharomyces pombe. The hotspot activity of the palindrome, but not the basal level of recombination, was abolished by a rad50 deletion, by a rad50S "separation of function" mutation, or by a rad32-D25A mutation in the nuclease domain of the Rad32 protein, an Mre11 homolog. We propose that upon extrusion of the palindrome the Rad50.Rad32 nuclease complex recognizes and cleaves the secondary structure thus formed and generates a recombinogenic break in the DNA.     

Meiotic localization of Mre11 and Rad50 in wild type, <Emphasis Type="Italic">spo11-1</Emphasis>, and MRN complex mutants of <Emphasis Type="Italic">Coprinus cinereus</Emphasis>

The Mre11-Rad50-Nbs1 (MRN) complex is required for numerous cellular processes that involve interactions with DNA double-strand breaks. For the majority of these processes, the MRN complex is thought to act as a unit, with each protein aiding the activity of the others. We have examined the relationship between Mre11 and Rad50 during meiosis in the basidiomycete Coprinus cinereus (Coprinopsis cinerea), investigating to what extent activities of Mre11 and Rad50 are interdependent. We showed that mre11-1 is epistatic to rad50-1 with respect to the time of meiotic arrest, indicating that Mre11 activity facilitates the diffuse diplotene arrest of rad50 mutants. Anti-Mre11 and anti-Rad50 antibodies were used to examine MRN complex localization in a wild-type strain and in spo11, mre11, and rad50 mutants. In wild type, numbers of Mre11 and Rad50 foci peaked at time points corresponding to leptotene and early zygotene. In the spo11-1 mutant, which is defective in meiotic double-strand break formation, foci accumulated throughout prophase I. Of seven MRN mutants examined, only two rad50 strains exhibited Mre11 and Rad50 foci that localized to chromatin, although Mre11 protein was found in the cell for all of them. Analysis of predicted mutant structures showed that stable localization of Mre11 and Rad50 does not depend upon a wild-type hook-proximal coiled coil, but does require the presence of the Rad50 ATPase/adenylate cyclase domains. We found that Mre11 and Rad50 were interdependent for binding to meiotic chromosomes. However, the majority of foci observed apparently contained only one of the two proteins. Independent Mre11 and Rad50 foci might indicate disassociation of the complex during meiosis or could reflect independent structural roles for the two proteins in meiotic chromatin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Saccharomyces cerevisiae mre11(ts) allele confers a separation of DNA repair and telomere maintenance functions

The yeast Mre11 protein participates in important cellular functions such as DNA repair and telomere maintenance. Analysis of structure-function relationships of Mre11 has led to identification of several separation-of-function mutations as well as N- and C-terminal domains essential for Mre11 meiotic and mitotic activities. Previous studies have established that there is a strong correlation between Mre11 DNA repair and telomere maintenance functions and that Mre11-Rad50-Xrs2 complex formation appears to be essential for both of these activities. Here we report that the mre11(ts) allele, previously shown to cause temperature-dependent defects in DNA repair and meiosis, confers a temperature-independent telomere shortening, indicating that mre11(ts) is a separation-of-function mutation with respect to DNA repair and telomere maintenance. In a yeast two-hybrid system, Mre11(ts) fails to form a homodimer or interact with Rad50 and Xrs2 irrespective of experimental temperatures. These observations collectively suggest that the Pro(162)Ser substitution in Mre11(ts) confers a novel separation of Mre11 mitotic functions. Moreover, we observed that while overexpression of the 5'-3' exonuclease gene EXO1 partially complements the MMS sensitivity of mre11, rad50, and xrs2 null mutants, it has no effect on telomere shortening in these strains. This result provides additional evidence on possible involvement of distinctive mechanisms in DNA repair and telomere maintenance by the Mre11-Rad50-Xrs2 complex.     

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.     

Rad50 Zinc Hook Is Important for the Mre11 Complex to Bind Chromosomal DNA Double-stranded Breaks and Initiate Various DNA Damage Responses

The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn(2+) promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.     

Mammalian Rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.     

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.     

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.