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Events upstream AID-mediated switch lesion are not affected in Nbs1deltaB/deltaB Atm -/- VAV B cells

Flow cytometric analysis of haematopoietic tissues

PDF file - 470KB, All supplementary figures including tumor histology, RAD50 immunohistochemistry, candidate mutations and their clonality, and supporting yeast and MEF experimental data.

PDF file - 2461KB, A complete listing of somatic mutations and rearrangements detected as well as targeted genes, sequences, MRN complex mutation frequency, and strains utilized.

Sμ-Sγ1 junctions

PDF file - 321KB, A detailed description of additional methods utilized (and associated references) for sequencing and analysis as well as an explanatory note regarding the TM pathway in yeast.

The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATMflox) was crossed to hypomorphic NBS1 mutants (Nbs1ΔB/ΔB mice). Nbs1ΔB/ΔB Atm−/− hematopoietic cells derived by crossing to vavcre were viable in vivo. Nbs1ΔB/ΔB Atm−/− VAV mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress.Implications: A novel mouse model was developed, by combining a vavcre-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis. Mol Cancer Res; 14(2); 185–95. ©2015 AACR.

Supplementary Figures 1-5, Table 1 from DNA Damage Signaling in Hematopoietic Cells: A Role for Mre11 Complex Repair of Topoisomerase Lesions

The Mre11 complex promotes DNA double-strand break repair and regulates DNA damage signaling via activation of the ataxia-telangiectasia mutated (ATM) kinase. The hypermorphic Rad50S allele encodes a variant of Rad50, a member of the Mre11 complex. Cells expressing Rad50S experience constitutive ATM activation, which leads to precipitous apoptotic attrition in hematopoietic cells. In this study, we show that ATM activation by the Rad50S-containing Mre11 complex enhances the proliferation of LSK cells, a population consisting of hematopoietic stem cells and multipotent progenitor cells. In Rad50S/S mice, enhanced LSK proliferation triggers apoptotic attrition. This phenotype is mitigated when Rad50S/S is combined with mutations that alter either LSK cell quiescence (myeloid elf-1–like factor/ELF4–deficient mice) or hematopoietic differentiation (p21- and p27-deficient mice), indicating that the LSK population is a primary target of Rad50S pathology. We show that cells from Rad50S/S mice are hypersensitive to camptothecin, a topoisomerase I inhibitor that causes DNA damage primarily during DNA replication. On this basis, we propose that apoptotic attrition of Rad50S/S hematopoietic cells results from enhanced proliferation in the context of topoisomerase-associated DNA damage. Impairment of apoptosis in Rad50S/S mice promotes hematopoietic malignancy, suggesting that primitive hematopoietic cells serve as a reservoir of potentially oncogenic lesions in Rad50S/S mice. These data provide compelling evidence that the Mre11 complex plays a role in the metabolism of topoisomerase lesions in mammals, and further suggest that such lesions can accumulate in primitive hematopoietic cells and confer significant oncogenic potential. [Cancer Res 2008;68(7):2186–93]

We derived a mouse model in which a mutant form of Nbn/Nbs1(mid8) (hereafter Nbn(mid8)) exhibits severely impaired binding to the Mre11−Rad50 core of the Mre11 complex. The Nbn(mid8) allele was expressed exclusively in hematopoietic lineages (in Nbn(−/mid8vav) mice). Unlike Nbn(flox/floxvav) mice with Nbn deficiency in the bone marrow, Nbn(−/mid8vav) mice were viable. Nbn(−/mid8vav) mice hematopoiesis was profoundly defective, exhibiting reduced cellularity of thymus and bone marrow, and stage-specific blockage of B cell development. Within 6 mo, Nbn(−/mid8) mice developed highly penetrant T cell leukemias. Nbn(−/mid8vav) leukemias recapitulated mutational features of human T cell acute lymphoblastic leukemia (T-ALL), containing mutations in NOTCH1, TP53, BCL6, BCOR, and IKZF1, suggesting that Nbn(mid8) mice may provide a venue to examine the relationship between the Mre11 complex and oncogene activation in the hematopoietic compartment. Genomic analysis of Nbn(−/mid8vav) malignancies showed focal amplification of 9qA2, causing overexpression of MRE11 and CHK1. We propose that overexpression of MRE11 compensates for the metastable Mre11−Nbn(mid8) interaction, and that selective pressure for overexpression reflects the essential role of Nbn in promoting assembly and activity of the Mre11 complex.

Defects in the DNA damage repair system result in increased genomic instability and have recently been implicated as being drivers of tumorigenesis in both familial and sporadic breast cancers. To maintain genomic integrity, cells have a DNA damage response (DDR) mechanism that functions to repair damaged DNA efficiently and commits cells to death if damage is irreparable. Failure of this mechanism results in genomic instability and cancer predisposition. Widespread chromosomal instability is a characteristic feature of Triple-Negative Breast Cancer (TNBC), making it difficult to decipher between genes that drive cancer development from those that play a bystander role. Little is known about what gives rise to the extensive genomic instability of TNBC, and presents a major deficit in our scientific and clinical knowledge. Oncogene induced hyper-proliferation results in replication-associated double strand breaks (DSBs) that engage an Mre11-Rad50-Nbs1 complex-dependent DDR. Classically, the oncogene induced DDR is believed to suppress tumorigenesis due to downstream activation of p53. Using genetically engineered primary mammary epithelial cell models, we demonstrate p53-independent effects of the Mre11-dependent DDR in suppressing proliferation and DNA damage induced by diverse oncogenic drivers. Single cell whole genome sequencing in Her2/Neu expressing primary mammary epithelial cells reveals a landscape of stochastic copy number aberrations induced by oncogenic stress that becomes enriched for a genomic scar pattern of larger-size deletions in cells with Mre11 hypomorphism. We identify Mre11 pathway hypomorphism in a subset of basal-like breast cancers (BLBC), which confers vulnerability to specific DNA damaging agents and DDR inhibitors in murine models of p53-deficient BLBC. Thus, assessing the functional status of the Mre11-dependent DDR pathway in p53-mutant breast cancers may provide an opportunity for therapeutic exploitation. Citation Format: Fagan-Solis KD, Simpson DA, Kapustina M, Martelotto L, Reis-Filho JS, Petrini JH, Gupta GP. A p53-independent DNA damage response that regulates breast cancer phenotypes [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-06-08.

Telomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the subtelomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA-containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations., Long non coding RNA TERRA transcripts can form R-loops at chromosome ends. Here, the authors reveal a role for the helicase RTEL in affecting TERRA levels and localization.

Summary The MRE11-RAD50-NBS1 complex plays a central role in response to DNA double-strand breaks. Here, we identify a patient with bone marrow failure and developmental defects caused by biallelic RAD50 mutations. One of the mutations creates a null allele, whereas the other (RAD50E1035Δ) leads to the loss of a single residue in the heptad repeats within the RAD50 coiled-coil domain. This mutation represents a human RAD50 separation-of-function mutation that impairs DNA repair, DNA replication, and DNA end resection without affecting ATM-dependent DNA damage response. Purified recombinant proteins indicate that RAD50E1035Δ impairs MRE11 nuclease activity. The corresponding mutation in Saccharomyces cerevisiae causes severe thermosensitive defects in both DNA repair and Tel1ATM-dependent signaling. These findings demonstrate that a minor heptad break in the RAD50 coiled coil suffices to impede MRE11 complex functions in human and yeast. Furthermore, these results emphasize the importance of the RAD50 coiled coil to regulate MRE11-dependent DNA end resection in humans., Graphical Abstract, Highlights • A human RAD50 mutant impairs DNA repair but not ATM-dependent DDR • A single amino acid deletion disrupts the coiled-coil structure of RAD50 • A Rad50 mutant yeast strain exhibits an extremely severe thermosensitive phenotype, Chansel-Da Cruz et al. provide evidence that a mutation in the human RAD50 coiled coil located away from the MRE11 interaction interface uncouples the signaling function of the MRN complex from its broken DNA end processing activities. This demonstrates the crucial role of the RAD50 coiled coil in the MRE11 complex regulation.

Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism, or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism, or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harboring mutations that impair their ability to correctly repair the DNA. Tumor predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene–environment interactions (GxE) influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer, will allow the design of novel and effective preventive and therapeutic strategies.

The Rad50 hook interface is crucial for assembly and various functions of the Mre11 complex. Previous analyses suggested that Rad50 molecules interact within (intracomplex) or between (intercomplex) dimeric complexes. In this study, we determined the structure of the human Rad50 hook and coiled-coil domains. The data suggest that the predominant structure is the intracomplex, in which the two parallel coiled coils proximal to the hook form a rod shape, and that a novel interface within the coiled-coil domains of Rad50 stabilizes the interaction of Rad50 protomers in the dimeric assembly. In yeast, removal of the coiled-coil interface compromised Tel1 activation without affecting DNA repair, while simultaneous disruption of that interface and the hook phenocopied a null mutation. The results demonstrate that the hook and coiled-coil interfaces coordinately promote intracomplex assembly and define the intracomplex as the functional form of the Mre11 complex.

Summary: The Mre11 complex (Mre11, Rad50, and Nbs1) is integral to both DNA repair and ataxia telangiectasia mutated (ATM)-dependent DNA damage signaling. All three Mre11 complex components are essential for viability at the cellular and organismal levels. To delineate essential and non-essential Mre11 complex functions that are mediated by Nbs1, we used TALEN-based genome editing to derive Nbs1 mutant mice (Nbs1mid mice), which harbor mutations in the Mre11 interaction domain of Nbs1. Nbs1mid alleles that abolished interaction were incompatible with viability. Conversely, a 108-amino-acid Nbs1 fragment comprising the Mre11 interface was sufficient to rescue viability and ATM activation in cultured cells and support differentiation of hematopoietic cells in vivo. These data indicate that the essential role of Nbs1 is via its interaction with Mre11 and that most of the Nbs1 protein is dispensable for Mre11 complex functions and suggest that Mre11 and Rad50 directly activate ATM. : Kim et al. find that Nbs1 promotes the proper assembly and localization of a complex containing Mre11 and Rad50. Nbs1-mediated assembly is required for the function of the complex, and a 108-amino-acid Nbs1 fragment containing the Mre11 interaction domain is sufficient for this essential role. Keywords: Nbs1mid mutants, Mre11-Nbs1 interface, ATM activation, Mre11 complex assembly

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells., Author summary A complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair.

Saccharomyces cerevisiae Tel1 is the ortholog of human ATM kinase and initiates a cell cycle checkpoint in response to dsDNA breaks (DSBs). Tel1(ATM) kinase is activated synergistically by naked dsDNA and the Mre11-Rad50-Xrs2(NBS1) complex (MRX). A multisubunit protein complex, which is related to human shelterin, protects telomeres from being recognized as DSBs, thereby preventing a Tel1(ATM) checkpoint response. However, at very short telomeres, Tel1(ATM) can be recruited and activated by the MRX complex, resulting in telomere elongation. Conversely, at long telomeres, Rap1-interacting-factor 2 (Rif2) is instrumental in suppressing Tel1 activity. Here, using an in vitro reconstituted Tel1 kinase activation assay, we show that Rif2 inhibits MRX-dependent Tel1 kinase activity. Rif2 discharges the ATP-bound form of Rad50, which is essential for all MRX-dependent activities. This conclusion is further strengthened by experiments with a Rad50 allosteric ATPase mutant that maps outside the conserved ATP binding pocket. We propose a model in which Rif2 attenuates Tel1 activity at telomeres by acting directly on Rad50 and discharging its activated ATP-bound state, thereby rendering the MRX complex incompetent for Tel1 activation. These findings expand our understanding of the mechanism by which Rif2 controls telomere length.

We derived a mouse model in which a mutant form of Nbs1 (Nbs1mid8) exhibits severely impaired binding to the Mre11-Rad50 core of the Mre11 complex. TheNbs1mid8allele was expressed exclusively in hematopoietic lineages (inNbs1-/mid8vavmice). UnlikeNbs1flox/floxvavmice, which are Nbs1 deficient in the bone marrow,Nbs1-/mid8vavmice were viable.Nbs1-/mid8vavhematopoiesis was profoundly defective, exhibiting reduced cellularity of thymus and bone marrow, and stage specific blockage of B cell development. Within six months,Nbs1-/mid8mice developed highly penetrant T cell leukemias.Nbs1-/mid8vavleukemias recapitulated mutational features of human T-ALL, containing mutations inNotch1, Trp53, Bcl6, Bcor, andIkzf1, suggesting thatNbs1mid8mice may provide a venue to examine the relationship between the Mre11 complex and oncogene activation in the hematopoietic compartment. Genomic analysis ofNbs1-/mid8vavmalignancies showed focal amplification of 9qA2, causing overexpression ofMRE11andCHK1. We propose that overexpression compensates for the meta-stable Mre11-Nbs1mid8interaction, and that selection pressure for overexpression reflects the essential role of Nbs1 in promoting assembly and activity of the Mre11 complex.

Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the two major pathways of DNA double strand break (DSB) repair and both are highly conserved from yeast to mammals. Nej1 has a role in DNA end tethering and the Mre11/Rad50/Xrs2 (MRX) complex is important for its recruitment to a DSB. Nej1 and Dna2- Sgs1 interact with the C-terminal end of Mre11, which also includes the region where Rad50 binds. Characterization of Nej1 in two rad50 mutants that alter the structural features of MRX showed that Nej1 inhibits Dna2 interactions with Mre11 and Sgs1. The work support a model whereby Nej1 binding to Mre11 provides a layer of regulation to repair pathway choice at the DSB.

Summary: The Mre11-Rad50-Nbs1 complex is a DNA double-strand break sensor that mediates a tumor-suppressive DNA damage response (DDR) in cells undergoing oncogenic stress, yet the mechanisms underlying this effect are poorly understood. Using a genetically inducible primary mammary epithelial cell model, we demonstrate that Mre11 suppresses proliferation and DNA damage induced by diverse oncogenic drivers through a p53-independent mechanism. Breast tumorigenesis models engineered to express a hypomorphic Mre11 allele exhibit increased levels of oncogene-induced DNA damage, R-loop accumulation, and chromosomal instability with a characteristic copy number loss phenotype. Mre11 complex dysfunction is identified in a subset of human triple-negative breast cancers and is associated with increased sensitivity to DNA-damaging therapy and inhibitors of ataxia telangiectasia and Rad3 related (ATR) and poly (ADP-ribose) polymerase (PARP). Thus, deficiencies in the Mre11-dependent DDR drive proliferation and genome instability patterns in p53-deficient breast cancers and represent an opportunity for therapeutic exploitation. : The origins of genome instability in cancer remain poorly understood. Fagan-Solis et al. reveal a p53-independent genome integrity checkpoint pathway mediated by Mre11 that protects against genome instability in breast cancer. Mre11 dysfunction in breast cancer models induces a genomic loss signature and vulnerability to PARP and ATR inhibitors. Keywords: breast cancer, genome instability, chromosomal instability, DNA damage response, oncogenic stress, Mre11, R loops, genomic scar, replication stress

The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATMflox) was crossed to hypomorphic NBS1 mutants (Nbs1ΔB/ΔB mice). Nbs1ΔB/ΔB Atm−/− hematopoietic cells derived by crossing to vavcre were viable in vivo. Nbs1ΔB/ΔB Atm−/− VAV mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress. Implications: A novel mouse model was developed, by combining a vavcre-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis. Mol Cancer Res; 14(2); 185–95. ©2015 AACR.

Phyllodes tumours (PTs) are breast fibroepithelial lesions that are graded based on histological criteria as benign, borderline or malignant. PTs may recur locally. Borderline PTs and malignant PTs may metastasize to distant sites. Breast fibroepithelial lesions, including PTs and fibroadenomas, are characterized by recurrent MED12 exon 2 somatic mutations. We sought to define the repertoire of somatic genetic alterations in PTs and whether these may assist in the differential diagnosis of these lesions. We collected 100 fibroadenomas, 40 benign PTs, 14 borderline PTs and 22 malignant PTs; six, six and 13 benign, borderline and malignant PTs, respectively, and their matched normal tissue, were subjected to targeted massively parallel sequencing (MPS) using the MSK-IMPACT sequencing assay. Recurrent MED12 mutations were found in 56% of PTs; in addition, mutations affecting cancer genes (eg TP53, RB1, SETD2 and EGFR) were exclusively detected in borderline and malignant PTs. We found a novel recurrent clonal hotspot mutation in the TERT promoter (-124 C>T) in 52% and TERT gene amplification in 4% of PTs. Laser capture microdissection revealed that these mutations were restricted to the mesenchymal component of PTs. Sequencing analysis of the entire cohort revealed that the frequency of TERT alterations increased from benign (18%) to borderline (57%) and to malignant PTs (68%; p < 0.01), and TERT alterations were associated with increased levels of TERT mRNA (p < 0.001). No TERT alterations were observed in fibroadenomas. An analysis of TERT promoter sequencing and gene amplification distinguished PTs from fibroadenomas with a sensitivity and a positive predictive value of 100% (CI 95.38-100%) and 100% (CI 85.86-100%), respectively, and a sensitivity and a negative predictive value of 39% (CI 28.65-51.36%) and 68% (CI 60.21-75.78%), respectively. Our results suggest that TERT alterations may drive the progression of PTs, and may assist in the differential diagnosis between PTs and fibroadenomas. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Despite intense efforts, the cure rates of childhood and adult solid tumors are not satisfactory. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. We have now found that the majority of childhood solid tumors, including rhabdoid tumors, neuroblastoma, medulloblastoma and Ewing sarcoma, express an active DNA transposasePGBD5that can promote site-specific genomic rearrangements in human cells. Using functional genetic approaches, we found that mouse and human cells deficient in non-homologous end joining (NHEJ) DNA repair cannot tolerate the expression of PGBD5. In a chemical screen of DNA damage signaling inhibitors, we identified AZD6738 as a specific sensitizer of PGBD5-dependent DNA damage and apoptosis. We found that expression of PGBD5, but not its nuclease activity-deficient mutant, was sufficient to induce hypersensitivity to AZD6738. Depletion of endogenous PGBD5 conferred resistance to AZD6738 in human tumor cells. PGBD5-expressing tumor cells accumulated unrepaired DNA damage in response to AZD6738 treatment, and underwent apoptosis in both dividing and G1 phase cells in the absence of immediate DNA replication stress. Accordingly, AZD6738 exhibited nanomolar potency against the majority of neuroblastoma, medulloblastoma, Ewing sarcoma and rhabdoid tumor cells tested, while sparing non-transformed human and mouse embryonic fibroblastsin vitro. Finally, treatment with AZD6738 induced apoptosis and regression of human neuroblastoma and medulloblastoma tumors engrafted in immunodeficient micein vivo. This effect was potentiated by combined treatment with cisplatin, including significant anti-tumor activity against patient-derived primary neuroblastoma xenografts. These findings delineate a therapeutically actionable synthetic dependency induced in PGBD5-expressing solid tumors.

Metastatic solid tumors are almost invariably fatal. Patients with disseminated small-cell cancers have a particularly unfavorable prognosis, with most succumbing to their disease within two years. Here, we report on the genetic and functional analysis of an outlier curative response of a patient with metastatic small-cell cancer to combined checkpoint kinase 1 (CHK1) inhibition and DNA-damaging chemotherapy. Whole-genome sequencing revealed a clonal hemizygous mutation in the Mre11 complex gene RAD50 that attenuated ATM signaling which in the context of CHK1 inhibition contributed, via synthetic lethality, to extreme sensitivity to irinotecan. As Mre11 mutations occur in a diversity of human tumors, the results suggest a tumor-specific combination therapy strategy in which checkpoint inhibition in combination with DNA-damaging chemotherapy is synthetically lethal in tumor cells but not normal cells with somatic mutations that impair Mre11 complex function. Significance: Strategies to effect deep and lasting responses to cancer therapy in patients with metastatic disease have remained difficult to attain, especially in early-phase clinical trials. Here, we present an in-depth genomic and functional genetic analysis identifying RAD50 hypomorphism as a contributing factor to a curative response to systemic combination therapy in a patient with recurrent, metastatic small-cell cancer. Cancer Discov; 4(9); 1014–21. ©2014 AACR. See related commentary by Peng et al., p. 988 This article is highlighted in the In This Issue feature, p. 973

DNA damage is considered a prime factor in multiple spinocerebellar neurodegenerative diseases; however, the DNA lesions underpinning disease etiology are unknown. Here we identify the endogenous accumulation of pathogenic topoisomerase-1-DNA cleavage complexes (Top1cc) in murine models of ataxia telangiectasia and spinocerebellar ataxia with axonal neuropathy 1. We also show that the defective DNA damage response factors in these two diseases cooperatively modulate Top1cc turnover in a non-epistatic and ATM kinase-independent manner. Furthermore, coincident neural inactivation of ATM and DNA single strand break repair factors including tyrosyl-DNA phosphodiesterase-1 or XRCC1 result in increased Top1cc formation and excessive DNA damage and neurodevelopmental defects. Importantly, direct topoisomerase-1 poisoning to elevate Top1cc levels phenocopies the neuropathology of the mouse models above. Our study identifies a critical endogenous pathogenic lesion associated with neurodegenerative syndromes arising from DNA repair deficiency, indicating the essential role that genome integrity plays in preventing disease in the nervous system.

SUMMARY Non-homologous end joining (NHEJ) and homologous recombination (HR) are the two major pathways of DNA double-strand break (DSB) repair and both are highly conserved from yeast to mammals. Nej1 has a role in DNA end-tethering at a DSB, and the Mre11/Rad50/Xrs2 (MRX) complex is important for its recruitment to the break. Nej1 and Dna2-Sgs1 interact with the C-terminal end of Mre11, which also includes the region where Rad50 binds. By characterizing the functionality of Nej1 in two rad50 mutants, which alter the structural features of MRX, we demonstrate that Nej1 inhibits the binding of Dna2 to Mre11 and Sgs1. Nej1 interactions with Mre11 promote tethering and inhibit hyper-resection, and when these events are compromised, large deletions develop at a DSB. The work indicates that Nej1 provides a layer of regulation to repair pathway choice and is consistent with its role in NHEJ., Graphical Abstract, In Brief Mojumdar et al. characterize the role of Nej1 during double-strand break repair. They show Nej1 promotes non-homologous end joining (NHEJ) by tethering the broken DNA ends and by inhibiting hyper-resection mediated by Dna2-Sgs1.

Squamous cell carcinoma-related oncogene (SCCRO, also known as DCUN1D1) is a component of the E3 for neddylation. As such, DCUN1D1 regulates the neddylation of cullin family members. Targeted inactivation of DCUN1D1 in mice results in male-specific infertility. Infertility in DCUN1D1-/- mice is secondary to primary defects in spermatogenesis. Time-dam experiments mapped the onset of the defect in spermatogenesis to 5.5 to 6 weeks of age, which temporally corresponds to defects in spermiogenesis. Although the first round of spermatogenesis progressed normally, the number of spermatozoa released into the seminiferous lumen and epididymis of DCUN1D1-/- mice was significantly reduced. Spermatozoa in DCUN1D1-/- mice had multiple abnormalities, including globozoospermia, macrocephaly, and multiple flagella. Many of the malformed spermatozoa in DCUN1D1-/- mice were multinucleated, with supernumerary and malpositioned centrioles, suggesting a defect in the resolution of intercellular bridges. The onset of the defect in spermatogenesis in DCUN1D1-/- mice corresponds to an increase in DCUN1D1 expression observed during normal spermatogenesis. Moreover, consistent with its known function as a component of the E3 in neddylation, the pattern of DCUN1D1 expression temporally correlates with an increase in the neddylated cullin fraction and stage-specific increases in the total ubiquitinated protein pool in wild-type mice. Levels of neddylated Cul3 were decreased in DCUN1D1-/- mice, and ubiquitinated proteins did not accumulate during the stages in which DCUN1D1 expression peaks during spermatogenesis in wild-type mice. Combined, these findings suggest that DCUN1D1-/- mice fail to release mature spermatozoa into the seminiferous lumen, possibly due to unresolved intercellular bridges. Furthermore, the effects of DCUN1D1 on spermatogenesis likely involve its regulation of cullin-RING-ligase (CRL)-type ubiquitin E3 activity during spermiogenesis through its role in promoting Cul3 neddylation. The specific CRLs required for spermiogenesis and their protein targets require identification.

The DNA damage response (DDR) is activated by oncogenic stress, but the mechanisms by which this occurs, and the particular DDR functions that constitute barriers to tumorigenesis, remain unclear. We established a mouse model of sporadic onco-gene-driven breast tumorigenesis in a series of mutant mouse strains with specific DDR deficiencies to reveal a role for the Mre11 complex in the response to oncogene activation. We demonstrate that an Mre11-mediated DDR restrains mammary hyperplasia by effecting an oncogene-induced G2 arrest. Impairment of Mre11 complex functions promotes the progression of mammary hyperplasias into invasive and metastatic breast cancers, which are often associated with secondary inactivation of the Ink4a-Arf (CDKN2a) locus. These findings provide insight into the mechanism of DDR engagement by activated oncogenes and highlight genetic interactions between the DDR and Ink4a-Arf pathways in suppression of oncogene-driven tumorigenesis and metastasis.

SummarySingle-ended double-strand breaks (DSBs) are a common form of spontaneous DNA break, generated when the replisome encounters a discontinuity in the DNA template. Given their prevalence, understanding the mechanisms governing the fate(s) of single-ended DSBs is important. We describe the influence of the Ku heterodimer and Mre11 nuclease activity on processing of single-ended DSBs. Separation-of-function alleles of yku70 were derived that phenocopy Ku deficiency with respect to single-ended DSBs but remain proficient for NHEJ. The Ku mutants fail to regulate Exo1 activity, and bypass the requirement for Mre11 nuclease activity in the repair of camptothecin-induced single-ended DSBs. Ku mutants exhibited reduced affinity for DNA ends, manifest as both reduced end engagement and enhanced probability of diffusing inward on linear DNA. This study reveals an interplay between Ku and Mre11 in the metabolism of single-ended DSBs that is distinct from repair pathway choice at double-ended DSBs.

Multiple myeloma is a plasma cell neoplasm with an extremely variable clinical course. Animal models are needed to better understand its pathophysiology and for preclinical testing of potential therapeutic agents. Hematopoietic cells expressing the hypermorphic Rad50s allele show hematopoietic failure, which can be mitigated by the lack of a transcription factor, Mef/Elf4. However, we find that 70% of Mef−/−Rad50s/s mice die from multiple myeloma or other plasma cell neoplasms. These mice initially show an abnormal plasma cell proliferation and monoclonal protein production, and then develop anemia and a decreased bone mineral density. Tumor cells can be serially transplanted and according to array CGH and whole exome sequencing, the pathogenesis of plasma cell neoplasms in these mice is not linked to activation of a specific oncogene, or inactivation of a specific tumor suppressor. This model recapitulates the systemic manifestations of human plasma cell neoplasms, and implicates cooperativity between the Rad50s and Mef/Elf4 pathways in initiating myelomagenic mutations that promote plasma cell transformation.

// Kasthuri Kannan 1,4 , Akiko Inagaki 2 , Joachim Silber 1,4 , Daniel Gorovets 1,4 , Jianan Zhang 1,4 , Edward R. Kastenhuber 1,4 , Adriana Heguy 4 , John H. Petrini 2 , Timothy A. Chan 3,4 , and Jason T. Huse 1,4 1 Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 2 Department of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 3 Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 4 Department of Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Correspondence: Jason T. Huse, email: // Keywords : glioma, astrocytoma, IDH, ATRX, whole-exome sequencing Received : October 01, 2012, Accepted : October 09, 2012, Published : October 11, 2012 Abstract The molecular foundations of lower-grade gliomas (LGGs)—astrocytoma, oligodendroglioma, and oligoastrocytoma—remain less well characterized than those of their fully malignant counterpart, glioblastoma. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) likely represent initiating pathogenic events. However, while IDH mutations appear to dramatically alter cellular epigenomic landscapes, definitive downstream transformative mechanisms have not been characterized. It remains likely, therefore, that additional genomic abnormalities collaborate with IDH mutation to drive oncogenesis in LGG. We performed whole exome sequencing in 4 LGGs, followed by focused resequencing in an additional 28, and found a high incidence of mutations in the ATRX gene (α thalassemia/mental retardation syndrome X-linked). ATRX forms a core component of a chromatin remodeling complex active in telomere biology. Mutations in ATRX have been identified in multiple tumor types and appear to cause alternative lengthening of telomeres (ALT), a presumed precursor to genomic instability. In our samples, ATRX mutation was entirely restricted to IDH-mutant tumors, closely correlated with TP53 mutation and astrocytic differentiation, and mutually exclusive with 1p/19q codeletion, the molecular hallmark of oligodendroglioma. Moreover, ATRX mutation was highly enriched in tumors of so-called early progenitor-like transcriptional subclass (~85%), which our prior work has linked to specific cells of origin in the forebrain subventricular zone. Finally, ATRX mutation correlated with ALT, providing a mechanistic link to genomic instability. In summary, our findings both identify ATRX mutation as a defining molecular determinant for a large subset of IDH-mutant gliomas and have direct implications on pathogenic mechanisms across the wide spectrum of LGGs.

The maintenance of genome stability depends on the DNA damage response (DDR), which is a functional network comprising signal transduction, cell cycle regulation and DNA repair. The metabolism of DNA double-strand breaks governed by the DDR is important for preventing genomic alterations and sporadic cancers, and hereditary defects in this response cause debilitating human pathologies, including developmental defects and cancer. The MRE11 complex, composed of the meiotic recombination 11 (MRE11), RAD50 and Nijmegen breakage syndrome 1 (NBS1; also known as nibrin) proteins is central to the DDR, and recent insights into its structure and function have been gained from in vitro structural analysis and studies of animal models in which the DDR response is deficient.

Chromosomal rearrangements and copy number aberrations are a major mechanism for driver gene alterations in breast cancer. However, the origins of chromosomal instability during breast tumorigenesis are poorly understood. We have recently shown that oncogene expression in normal mammary epithelial cells induces replication stress and a DNA damage response (DDR). The double-strand break sensor Mre11 is required for the oncogene-induced DDR, and suppression of Mre11 is sufficient to promote the development of Her2/Neu-driven breast cancer in a transgenic mouse model. Here we have applied single-cell genomics to primary mammary epithelial cells from this mouse model and demonstrate a profound effect of oncogene expression on chromosomal instability in preneoplastic cells, which is further augmented in Mre11-mutant cells. Her2/Neu-induced chromosomal breakpoints are highly enriched in the vicinity of large genes, suggesting a possible role for gene transcription and/or R-loops in the origin of oncogene-induced genomic fragility. Mre11 hypomorphism does not affect the number of chromosomal breakpoints but alters the genomic aberration signature to one that is over-represented by larger-size deletions. These findings indicate that chromosomal instability is an early event during Her2/Neu-initiated breast neoplasia. Furthermore, DDR perturbation—in the form of Mre11 hypomorphism—is not required for oncogene-induced genomic instability but rather alters the pattern of observed copy number aberrations and promotes the proliferative expansion of genomically unstable clones. Thus, single-cell genomics applied to murine models of breast preneoplasia reveals insights into the origins of chromosomal instability in breast cancer that may inform novel strategies to prognosticate or prevent the progression of high-risk premalignant breast lesions. Citation Format: Katerina Fagan-Solis, Dennis A. Simpson, Luciano Martelotto, Jorge S. Reis-Filho, John H. Petrini, Gaorav P. Gupta. Early onset of chromosomal instability in breast preneoplasia detected by single-cell genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3376.

Nijmegen breakage syndrome (NBS) is characterized by genome instability and cancer predisposition. NBS patients contain a mutation in the NBS1 gene, which encodes the NBS1 component of the DNA double-strand break (DSB) response complex MRE11/RAD50/NBS1. To investigate the NBS phenotype in more detail, we combined the mouse mimic of the most common patient mutation (Nbs1(Delta B/Delta B)) with a Rad54 null mutation, which diminishes homologous recombination. Double mutant cells were particularly sensitive to treatments that cause single strand breaks (SSBs), presumably because these SSBs can be converted into detrimental DSBs upon passage of a replication fork. The persistent presence of nuclear RAD51 foci and increased levels of chromatid type breaks in metaphase spreads indicated that replication-associated DSBs are repaired inefficiently in the double mutant cells. We conclude that Nbs1 and Rad54 function cooperatively, but in separate pathways to counteract this type of DNA damage and discuss mechanistic implications of these findings. (C) 2009 Elsevier B.V. All rights reserved.

In this study, we examine the telomeric functions of the mammalian Mre11 complex by using hypomorphic Mre11 and Nbs1 mutants (Mre11(ATLD1/ATLD1) and Nbs1(Delta)(B/)(DeltaB), respectively). No telomere shortening was observed in Mre11(ATLD1/ATLD1) cells after extensive passage through culture, and the rate of telomere shortening in telomerase-deficient (Tert(Delta)(/)(Delta)) Mre11(ATLD1/ATLD1) cells was the same as that in Tert(Delta)(/)(Delta) alone. Although telomeres from late-passage Mre11(ATLD1/ATLD1) Tert(Delta)(/)(Delta) cells were as short as those from Tert(Delta)(/)(Delta), the incidence of telomere fusions was reduced. This effect on fusions was also evident upon acute telomere dysfunction in Mre11(ATLD1/ATLD1) and Nbs1(Delta)(B/)(DeltaB) cells rendered Trf2 deficient by cre-mediated TRF2 inactivation than in wild-type cells. The residual fusions formed in Mre11 complex mutant cells exhibited a strong tendency toward chromatid fusions, with an almost complete bias for fusion of telomeres replicated by the leading strand. Finally, the response to acute telomere dysfunction was strongly impaired by Mre11 complex hypomorphism, as the formation of telomere dysfunction-induced DNA damage foci was reduced in both cre-infected Mre11(ATLD1/ATLD1) Trf2(F/)(Delta) and Nbs1(Delta)(B/)(DeltaB) Trf2(F/F) cells. These data indicate that the Mre11 complex influences the cellular response to telomere dysfunction, reminiscent of its influence on the response to interstitial DNA breaks, and suggest that it may promote telomeric DNA end processing during DNA replication.

C57BL/6J mice carrying the Min allele of Adenomatous polyposis coli (Apc) develop numerous adenomas along the entire length of the intestine and consequently die at an early age. This short lifespan would prevent the accumulation of somatic genetic mutations or epigenetic alterations necessary for tumor progression. To overcome this limitation, we generated F1 ApcMin/+ hybrids by crossing C57BR/cdcJ and SWR/J females to C57BL/6J ApcMin/+ males. These hybrids developed few intestinal tumors and often lived longer than 1 year. Many of the tumors (24-87%) were invasive adenocarcinomas, in which neoplastic tissue penetrated through the muscle wall into the mesentery. In a few cases (3%), lesions metastasized by extension to regional lymph nodes. The development of these familial cancers does not require chromosomal gains or losses, a high level of microsatellite instability, or the presence of Helicobacter. To test whether genetic instability might accelerate tumor progression, we generated ApcMin/+ mice homozygous for the hypomorphic allele of the Nijmegen breakage syndrome gene (Nbs1ΔB) and also treated ApcMin/+ mice with a strong somatic mutagen. These imposed genetic instabilities did not reduce the time required for cancers to form nor increase the percentage of cancers nor drive progression to the point of distant metastasis. In summary, we have found that the ApcMin/+ mouse model for familial intestinal cancer can develop frequent invasive cancers in the absence of overt genomic instability. Possible factors that promote invasion include age-dependent epigenetic changes, conservative somatic recombination, or direct effects of alleles in the F1 hybrid genetic background. [Cancer Res 2009;69(14):5768–75]

Genomic integrity is maintained via the concerted action of proteins that coordinate and control DNA replication and those that respond to DNA damage. The Mre11 complex is a mediator of the DNA damage response through its functions in DNA double strand break (DSB) sensing, checkpoint activation and recombinational DNA repair. The complex responds to mitotic and meiotic DSBs, and is also activated in cells experiencing DNA replication stress. The Mre11 complex's role in recombinational repair primarily concerns the promotion of homologous recombination (HR), but it is also implicated in non-homologous end joining (NHEJ)--a DSB repair mechanism prevalent in non-dividing cells. We recently characterized deletion of the Mre11 complex member, Rad50, in a number of postmitotic and proliferative tissues of the mouse. These studies indicated that the complex is dispensable in postmitotic tissues, but loss of Rad50 in proliferating cells resulted in accumulation of unrepaired, DNA replication-dependent lesions. The data suggest that the Mre11 complex is not a major contributor to NHEJ and support the interpretation that its role in recombinational DNA repair is largely restricted to dividing cells, in which repair involving sister chromatids predominates. An exception to this concept is manifest in previous work from our laboratory revealing that the mammalian Mre11 complex promotes meiotic DSB repair, an event involving recombination between sister chromatids of homologous chromosomes and taking place in cells not undergoing replication. Together these studies highlight the importance of cell cycle and cell type specific modulation of the Mre11 complex's repair activities in vivo.

Recent work has highlighted the importance of alternative, error-prone mechanisms for joining DNA double-strand breaks (DSB) in mammalian cells. These noncanonical, non-homologous end joining (NHEJ) pathways threaten genomic stability but remain poorly characterized. The RAG post-cleavage complex normally prevents V(D)J recombination-associated DSBs from accessing alternative NHEJ. Because the MRE11/RAD50/NBS1 complex localizes to RAG-mediated DSBs and possesses DNA end tethering, processing and joining activities, we asked whether it plays a role in the mechanism of alternative NHEJ, or participates in regulating access of DSBs to alternative repair pathways. We find that NBS1 is required for alternative NHEJ of hairpin coding ends, suppresses alternative NHEJ of signal ends, and promotes proper resolution of inversional recombination intermediates. These data demonstrate that the MRE11 complex functions at two distinct levels, regulating repair pathway choice (likely through enhancing the stability of DNA-end complexes) and participating in alternative NHEJ of coding ends.

Oligomeric assembly of Brca1 C-terminal (BRCT) domain-containing mediator proteins occurs at sites of DNA damage. However, the functional significance and regulation of such assemblies are not well understood. In this study, we defined the molecular mechanism of DNA-damage-induced oligomerization of the S. cerevisiae BRCT protein Rad9. Our data suggest that Rad9's tandem BRCT domain mediates Rad9 oligomerization via its interaction with its own Mec1/Tel1-phosphorylated SQ/TQ cluster domain (SCD). Rad53 activation is unaffected by mutations that impair Rad9 oligomerization, but checkpoint maintenance is lost, indicating that oligomerization is required to sustain checkpoint signaling. Once activated, Rad53 phosphorylates the Rad9 BRCT domain, which attenuates the BRCT-SCD interaction. Failure to phosphorylate the Rad9 BRCT results in cytologically visible Rad9 foci. This suggests a feedback loop wherein Rad53 activity and Rad9 oligomerization are regulated to tune the DNA-damage response.

Central to the DNA damage response (DDR) is the highly conserved Mre11 complex consisting of Mre11, Rad50 and Nbs1. The Mre11 complex acts as a sensor of DNA double-strand breaks (DSBs) and regulates the signal transduction cascades that are triggered following damage detection.(1) Rare human genetic instability syndromes such as Ataxia-telangiectasia (A-T) and Nijmegen Breakage Syndrome (NBS) have underscored the importance of the DSB response in the suppression of tumorigenesis, as well as other severe pathologies affecting the development of both the immune system and the central nervous system. Using murine models of the human diseases, we have investigated the role of the Mre11 complex, and other modulators of the DSB response, in tumor suppression.(2,3) We found that the checkpoint kinase Chk2 is crucial for the suppression of a diverse array of tumor types in Mre11 complex mutants and uncovered multiple roles for the Mre11 complex in apoptotic signaling in parallel to Chk2.(4,5).

The Mre11 complex functions in double-strand break (DSB) repair, meiotic recombination, and DNA damage checkpoint pathways. Sae2 deficiency has opposing effects on the Mre11 complex. On one hand, it appears to impair Mre11 nuclease function in DNA repair and meiotic DSB processing, and on the other, Sae2 deficiency activates Mre11-complex-dependent DNA-damage-signaling via the Tel1–Mre11 complex (TM) pathway. We demonstrate that SAE2 overexpression blocks the TM pathway, suggesting that Sae2 antagonizes Mre11-complex checkpoint functions. To understand how Sae2 regulates the Mre11 complex, we screened for sae2 alleles that behaved as the null with respect to Mre11-complex checkpoint functions, but left nuclease function intact. Phenotypic characterization of these sae2 alleles suggests that Sae2 functions as a multimer and influences the substrate specificity of the Mre11 nuclease. We show that Sae2 oligomerizes independently of DNA damage and that oligomerization is required for its regulatory influence on the Mre11 nuclease and checkpoint functions.

The MRE11 complex (MRE11, RAD50 and NBS1) and the ataxia-telangiectasia mutated (ATM) kinase function in the same DNA damage response pathway to effect cell cycle checkpoint activation and apoptosis. The functional interaction between the MRE11 complex and ATM has been proposed to require a conserved C-terminal domain of NBS1 for recruitment of ATM to sites of DNA damage. Human Nijmegen breakage syndrome (NBS) cells and those derived from multiple mouse models of NBS express a hypomorphic NBS1 allele that exhibits impaired ATM activity despite having an intact C-terminal domain. This indicates that the NBS1 C terminus is not sufficient for ATM function. We derived Nbs1(DeltaC/DeltaC) mice in which the C-terminal ATM interaction domain is deleted. Nbs1(DeltaC/DeltaC) cells exhibit intra-S-phase checkpoint defects, but are otherwise indistinguishable from wild-type cells with respect to other checkpoint functions, ionizing radiation sensitivity and chromosome stability. However, multiple tissues of Nbs1(DeltaC/DeltaC) mice showed a severe apoptotic defect, comparable to that of ATM- or CHK2-deficient animals. Analysis of p53 transcriptional targets and ATM substrates showed that, in contrast to the phenotype of Chk2(-/-) mice, NBS1(DeltaC) does not impair the induction of proapoptotic genes. Instead, the defects observed in Nbs1(DeltaC/DeltaC) result from impaired phosphorylation of ATM targets including SMC1 and the proapoptotic factor, BID.

Summary The Mre11 complex (consisting of MRE11, RAD50, and NBS1/Xrs2) is required for double-strand break (DSB) formation, processing, and checkpoint signaling during meiotic cell division in S. cerevisiae [1–8]. Whereas studies of Mre11 complex mutants in S. pombe and A. thaliana indicate that the complex has other essential meiotic roles [9–11], relatively little is known regarding the functions of the complex downstream of meiotic break formation and processing or its role in meiosis in higher eukaryotes. We analyzed meiotic events in mice harboring hypomorphic Mre11 and Nbs1 mutations which, unlike null mutants, support viability [12–16]. Our studies revealed defects in the temporal progression of meiotic prophase, incomplete and aberrant synapsis of homologous chromosomes, persistence of strand exchange proteins, and alterations in both the frequency and placement of MLH1 foci, a marker of crossovers. A unique sex-dependent effect on MLH1 foci and chiasmata numbers was observed: males exhibited an increase and females a decrease in recombination levels. Thus, our findings implicate the Mre11 complex in meiotic DNA repair and synapsis in mammals and indicate that the complex may contribute to the establishment of normal sex-specific differences in meiosis.

The MRE11 complex (MRE11, RAD50, and NBS1) is a central component of the DNA damage response, governing both double-strand break repair and DNA damage response signaling. To determine the functions of the MRE11 complex in the development and maintenance of oocytes, we analyzed ovarian phenotypes of mice harboring the hypomorphic Mre11ATLD1 allele. Mre11ATLD1/ATLD1 females exhibited premature oocyte elimination attributable to defects in homologous chromosome pairing and double-strand break repair during meiotic prophase. Other aspects of meiotic progression, including attachment of telomeres to the nuclear envelope and recruitment of RAD21L, a component of the meiotic cohesin complex to the synaptonemal complex, were normal. Unlike Dmc1−/− and Trp13Gt/Gt mice which exhibit comparable defects in double-strand break repair and oocyte depletion by 5 days post-partum, we found that oocyte attrition occurred by 12 weeks in Mre11ATLD1/ATLD1. Disruption of the oocyte checkpoint pathway governed by Chk2 gene further enhanced the survival of Mre11ATLD1/ATLD1 follicles. Together our data suggest that the MRE11 complex influences the elimination of oocytes with unrepaired meiotic double-strand breaks post-natally, in addition to its previously described role in double-strand break repair and homologous synapsis during female meiosis. Electronic supplementary material The online version of this article (doi:10.1007/s00412-015-0535-8) contains supplementary material, which is available to authorized users.

Genetic and cytologic data from Saccharomyces cerevisiae and mammals implicate the Mre11 complex, consisting of Mre11, Rad50, and Nbs1, as a sensor of DNA damage, and indicate that the complex influences the activity of ataxia-telangiectasia mutated (ATM) in the DNA damage response. Rad50S/S mice exhibit precipitous apoptotic attrition of hematopoietic cells. We generated ATM- and Chk2-deficient Rad50S/S mice and found that Rad50S/S cellular attrition was strongly ATM and Chk2 dependent. The hypomorphic Mre11ATLD1 and Nbs1ΔB alleles conferred similar rescue of Rad50S/S-dependent hematopoietic failure. These data indicate that the Mre11 complex activates an ATM–Chk2-dependent apoptotic pathway. We find that apoptosis and cell cycle checkpoint activation are parallel outcomes of the Mre11 complex–ATM pathway. Conversely, the Rad50S mutation mitigated several phenotypic features of ATM deficiency. We propose that the Rad50S allele is hypermorphic for DNA damage signaling, and that the resulting constitutive low-level activation of the DNA damage response accounts for the partial suppression of ATM deficiency in Rad50S/S Atm-/- mice.