The MRE11 GAR motif regulates DNA double-strand break processing and ATR activation

The MRE11/RAD50/NBS1 complex is the primary sensor rapidly recruited to DNA double-strand breaks (DSBs). MRE11 is known to be arginine methylated by PRMT1 within its glycine-arginine-rich (GAR) motif. In this study, we report a mouse knock-in allele of Mre11 that substitutes the arginines with lysines in the GAR motif and generates the MRE11(RK) protein devoid of methylated arginines. The Mre11(RK/RK) mice were hypersensitive to γ-irradiation (IR) and the cells from these mice displayed cell cycle checkpoint defects and chromosome instability. Moreover, the Mre11(RK/RK) MEFs exhibited ATR/CHK1 signaling defects and impairment in the recruitment of RPA and RAD51 to the damaged sites. The M(RK)RN complex formed and localized to the sites of DNA damage and normally activated the ATM pathway in response to IR. The M(RK)RN complex exhibited exonuclease and DNA-binding defects in vitro responsible for the impaired DNA end resection and ATR activation observed in vivo in response to IR. Our findings provide genetic evidence for the critical role of the MRE11 GAR motif in DSB repair, and demonstrate a mechanistic link between post-translational modifications at the MRE11 GAR motif and DSB processing, as well as the ATR/CHK1 checkpoint signaling.     

The GAR Motif of 53BP1 is Arginine Methylated by PRMT1 and is Necessary for 53BP1 DNA Binding Activity

The p53-binding protein 1 (53BP1) is rapidly recruited to sites of DNA double-strand breaks and forms characteristics nuclear foci, demonstrating its role in the early events of detection, signaling and repair of damaged DNA. 53BP1 contains a glycine arginine rich (GAR) motif of unknown function within its kinetochore binding domain. Herein, we show that the GAR motif of 53BP1 is arginine methylated by protein arginine methyltransferase 1 (PRMT1), the same methyltransferase that methylates MRE11. 53BP1 contains asymmetric dimethylarginines (aDMA) within cells, as detected with methylarginine-specific antibodies. Amino acid substitution of the arginines within the GAR motif of 53BP1 abrogated binding to single and double-stranded DNA, demonstrating that the GAR motif is required for DNA binding activity of 53BP1. Fibroblast cells treated with methylase inhibitors failed to relocalize 53BP1 to sites of DNA damage and formed few ?-H2AX foci, consistent with our previous data that MRE11 fails to relocalize to DNA damage sites in cells treated with methylase inhibitors. Our findings identify the GAR motif as a region required for 53BP1 DNA binding activity and is the site of methylation by PRMT1.     

MRE11 complex links RECQ5 helicase to sites of DNA damage

RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11–RAD50–NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3′→5′ exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repair.     

Methylation of MRE11 Regulates its Nuclear Compartmentalization

The cellular response to DNA damage includes the orderly recruitment of many proteincomplexes to DNA lesions. The MRE11-RAD50-NBS1 (MRN) complex is well knownto localize early to sites of DNA damage, but the post-translational modificationsrequired to mobilize it to DNA damage sites are poorly understood. Recently, we haveshown that MRE11 is arginine methylated in a C-terminal glycine-arginine rich (GAR)domain by protein arginine methyltransferase 1 (PRMT1). Arginine methylation isrequired for the exonuclease activity of MRE11 and the intra-S phase DNA damageresponse. Herein, we report that cells treated with methylase inhibitors failed to relocalizeMRE11 from PML nuclear bodies to sites of DNA damage and formed few ?-H2AX foci. We also demonstrate that PRMT1 is a component of PML nuclear bodieswhere it co-localizes with MRE11. Using cellular fractionation, we demonstrate thatmethylated MRE11 is predominantly associated with nuclear structures and that MRE11methylated arginines were required for this association. These results suggest thatMRE11 methylation regulates its association with nuclear structures such as PML nuclearbodies and sites of DNA damage.     

MRE11-RAD50-NBS1 and ATM function as co-mediators of TRF1 in telomere length control

Human telomeres are associated with ATM and the protein complex consisting of MRE11, RAD50 and NBS1 (MRN), which are central to maintaining genomic stability. Here we show that when targeted to telomeres, wild-type RAD50 downregulates telomeric association of TRF1, a negative regulator of telomere maintenance. TRF1 binding to telomeres is upregulated in cells deficient in NBS1 or under ATM inhibition. The TRF1 association with telomeres induced by ATM inhibition is abrogated in cells lacking MRE11 or NBS1, suggesting that MRN and ATM function in the same pathway controlling TRF1 binding to telomeres. The ability of TRF1 to interact with telomeric DNA in vitro is impaired by ATM-mediated phosphorylation. We propose that MRN is required for TRF1 phosphorylation by ATM and that such phosphorylation results in the release of TRF1 from telomeres, promoting telomerase access to the ends of telomeres.     

MRE11/RAD50 cleaves DNA in the AID/UNG-dependent pathway of immunoglobulin gene diversification

MRE11/RAD50/NBS1 (MRN) is a ubiquitous complex that participates in the response to DNA damage and in immunoglobulin (Ig) gene diversification. Ig gene diversification is initiated by deamination of cytosine to uracil, followed by removal of uracil to create an abasic (AP) site. We find that MRE11 associates specifically with rearranged Ig genes in hypermutating B cells, whereas APE1, the major AP-endonuclease in faithful base excision repair, does not. We show that purified, recombinant MRE11/RAD50 can cleave DNA at AP sites and that this AP-lyase activity is conserved from humans to Archaea. MRE11/RAD50 cleaves at AP sites within single-stranded regions of DNA, suggesting that at transcribed Ig genes, cleavage may be coordinated with deamination by AID and deglycosylation by UNG2 to produce single-strand breaks (SSBs) that undergo subsequent mutagenic repair and recombination. These results identify MRN with DNA cleavage in the AID-initiated pathway of Ig gene diversification.     

RAD50 and NBS1 form a stable complex functional in DNA binding and tethering

The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Previous data suggest that one important role for RMN in DSB repair is to provide a link between DNA ends. The striking architecture of the complex, a globular domain from which two extended coiled coils protrude, is essential for this function. Due to its DNA-binding activity, ability to form dimers and interact with both RAD50 and NBS1, MRE11 is considered to be crucial for formation and function of RMN. Here, we show the successful expression and purification of a stable complex containing only RAD50 and NBS1 (RN). The characteristic architecture of the complex was not affected by absence of MRE11. Although MRE11 is a DNA-binding protein it was not required for DNA binding per se or DNA-tethering activity of the complex. The stoichiometry of NBS1 in RMN and RN complexes was estimated by SFM-based volume analysis. These data show that in vitro, R, M and N form a variety of stable complexes with variable subunit composition and stoichiometry, which may be physiologically relevant in different aspects of RMN function.     

CDK targeting of NBS1 promotes DNA-end resection, replication restart and homologous recombination

The conserved MRE11–RAD50–NBS1 (MRN) complex is an important sensor of DNA double-strand breaks (DSBs) and facilitates DNA repair by homologous recombination (HR) and end joining. Here, we identify NBS1 as a target of cyclin-dependent kinase (CDK) phosphorylation. We show that NBS1 serine 432 phosphorylation occurs in the S, G2 and M phases of the cell cycle and requires CDK activity. This modification stimulates MRN-dependent conversion of DSBs into structures that are substrates for repair by HR. Impairment of NBS1 phosphorylation not only negatively affects DSB repair by HR, but also prevents resumption of DNA replication after replication-fork stalling. Thus, CDK-mediated NBS1 phosphorylation defines a molecular switch that controls the choice of repair mode for DSBs.     

The MRN complex promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

Homologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 and MRE11 are required for RAD51-dependent homologous recombination and phosphorylation of histone H2A following a DNA double strand break (DSB), but neither MRE11 nor RAD50 substantially influence DSB resection at a chromosome-internal locus. In addition, we reveal intrinsic separation-of-function between T. brucei RAD50 and MRE11, with only RAD50 suppressing DSB repair using donors with short stretches of homology at a subtelomeric locus, and only MRE11 directing DSB resection at the same locus. Finally, we show that loss of either MRE11 or RAD50 causes a greater diversity of expressed VSG variants following DSB repair. We conclude that MRN promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation.     

NBS1在DNA断裂损伤反应和维持端粒稳定中的作用

NBS1作为MRE11/RAD50/NBS1复合物的组分之一,是细胞应答DNA损伤的一个关键蛋白质,在DNA双链断裂修复和维持基因组稳定中发挥重要的作用。端粒是染色体末端由DNA重复序列和蛋白质构成的复合体,其独特结构与DNA双链断裂非常相似。最近几年的研究发现NBS1与端粒也有着十分密切的联系。综述了NBS1在DNA损伤反应中的作用,并探讨NBS1参与维持端粒稳定中的分子机制。     

Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting techniques. We show that cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways.     

Phospho-dependent interactions between NBS1 and MDC1 mediate chromatin retention of the MRN complex at sites of DNA damage

Mammalian cells respond to DNA double-strand breaks (DSBs) by recruiting DNA repair and cell-cycle checkpoint proteins to such sites. Central to these DNA damage response (DDR) events is the DNA damage mediator protein MDC1. MDC1 interacts with several DDR proteins, including the MRE11–RAD50–NBS1 (MRN) complex. Here, we show that MDC1 is phosphorylated on a cluster of conserved repeat motifs by casein kinase 2 (CK2). Moreover, we establish that this phosphorylation of MDC1 promotes direct, phosphorylation-dependent interactions with NBS1 in a manner that requires the closely apposed FHA and twin BRCT domains in the amino terminus of NBS1. Finally, we show that these CK2-targeted motifs in MDC1 are required to mediate NBS1 association with chromatin-flanking sites of unrepaired DSBs. These findings provide a molecular explanation for the MDC1–MRN interaction and yield insights into how MDC1 coordinates the focal assembly and activation of several DDR factors in response to DNA damage.     

DNA damage responses in Drosophila nbs mutants with reduced or altered NBS function

The MRN complex, composed of MRE11, RAD50 and NBS, plays important roles in responding to DNA double-strand breaks (DSBs). In metazoans, functional studies of genes encoding these proteins have been challenging because complete loss-of-function mutations are lethal at the organismal level and because NBS has multiple functions in DNA damage responses. To study functions of Drosophila NBS in DNA damage responses, we used a separation-of-function mutation that causes loss of the forkhead-associated (FHA) domain. Loss of the FHA domain resulted in hypersensitivity to ionizing radiation and defects in gap repair by homologous recombination, but had only a small effect on the DNA damage checkpoint response and did not impair DSB repair by end joining. We also found that heterozygosity for an nbs null mutation caused reduced gap repair and loss of the checkpoint response to low-dose irradiation. These findings shed light on possible sources of the cancer predisposition found in human carriers of NBN mutations.     

The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis

The repair of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. In higher eukaryotes, DNA DSBs are predominantly repaired by non-homologous end joining (NHEJ), but DNA ends can also be joined by an alternative error-prone mechanism termed microhomology-mediated end joining (MMEJ). In MMEJ, the repair of DNA breaks is mediated by annealing at regions of microhomology and is always associated with deletions at the break site. In budding yeast, the Mre11/Rad5/Xrs2 complex has been demonstrated to play a role in both classical NHEJ and MMEJ, but the involvement of the analogous MRE11/RAD50/NBS1 (MRN) complex in end joining in higher eukaryotes is less certain. Here we demonstrate that in Xenopus laevis egg extracts, the MRN complex is not required for classical DNA-PK-dependent NHEJ. However, the XMRN complex is necessary for resection-based end joining of mismatched DNA ends. This XMRN-dependent end joining process is independent of the core NHEJ components Ku70 and DNA-PK, occurs with delayed kinetics relative to classical NHEJ and brings about repair at sites of microhomology. These data indicate a role for the X. laevis MRN complex in MMEJ.     

RAD50/MRE11/NBS1 proteins in relation to tumour development and prognosis in patients with microsatellite stable colorectal cancer

RAD50/MRE11/NBS1 complex is essential for DNA double-strand break repair and for maintaining genomic integrity. In this study, we immunohistochemically examined MRE11, NBS1 and RAD50 expression in primary CRCs (n=208), the corresponding distant (n=41) and adjacent normal mucosa (n=130), and lymph node metastases (n=26), and investigated their clinicopathological significance in colorectal cancers (CRCs). We found that the intensity and percentage of MRE11 and NBS1 in primary CRCs were positively correlated with each other and with RAD50 (P<0.0001). Strong expression of MRE11, NBS1 or combined RAD50/MRE11/NBS1 was related to MSS, positive hMLH1 expression, earlier tumour stage (TNM stage I and II) and favourable survival (P<0.05). A high percentage of MRE11 expression was associated with less local recurrence and high apoptotic activity (P<0.05). In MSS CRCs, the expression of MRE11 and NBS1 was stronger than that in normal mucosa (P<0.05), and strong expression of NBS1 in primary tumour was related to favourable survival of patients in TNM stage I and II (univariate analysis: P=0.03; multivariate analysis: P=0.07). In MSI CRCs, neither MRE11 nor NBS1 expression showed differences among normal mucosa, primary tumour and metastasis, or among clinicopathological variables. In conclusion, RAD50/MRE11/NBS1 proteins interacted with each other, which had different clinicopathological significance in MSS and MSI CRCs, and further, each component of the complex might have additional roles. NBS1 might be a prognostic factor for patients with MSS tumour in TNM stage I and II.     

Constitutive phosphorylation of MDC1 physically links the MRE11-RAD50-NBS1 complex to damaged chromatin

The MRE11-RAD50-Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin.     

SIRT1 regulates the function of the Nijmegen breakage syndrome protein

MRE11-RAD50-NBS1 (MRN) is a conserved nuclease complex that exhibits properties of a DNA damage sensor and is critical in regulating cellular responses to DNA double-strand breaks. NBS1, which is mutated in the human genetic disease Nijmegen breakage syndrome, serves as the regulatory subunit of MRN. Phosphorylation of NBS1 by the ATM kinase is necessary for both activation of the S phase checkpoint and for efficient DNA damage repair response. Here, we report that NBS1 is an acetylated protein and that the acetylation level is tightly regulated by the SIRT1 deacetylase. SIRT1 associates with the MRN complex and, importantly, maintains NBS1 in a hypoacetylated state, which is required for ionizing radiation-induced NBS1 Ser343 phosphorylation. Our results demonstrate the presence of crosstalk between two different posttranslational modifications in NBS1 and strongly suggest that deacetylation of NBS1 by SIRT1 plays a key role in the dynamic regulation of the DNA damage response and in the maintenance of genomic stability.     

ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.     

A divalent FHA/BRCT‐binding mechanism couples the MRE11–RAD50–NBS1 complex to damaged chromatin

The MRE11–RAD50–NBS1 (MRN) complex accumulates at sites of DNA double‐strand breaks in large chromatin domains flanking the lesion site. The mechanism of MRN accumulation involves direct binding of the Nijmegen breakage syndrome 1 (NBS1) subunit to phosphorylated mediator of the DNA damage checkpoint 1 (MDC1), a large nuclear adaptor protein that interacts directly with phosphorylated H2AX. NBS1 contains an FHA domain and two BRCT domains at its amino terminus. Here, we show that both of these domains participate in the interaction with phosphorylated MDC1. Point mutations in key amino acid residues of either the FHA or the BRCT domains compromise the interaction with MDC1 and lead to defects in MRN accumulation at sites of DNA damage. Surprisingly, only mutation in the FHA domain, but not in the BRCT domains, yields a G2/M checkpoint defect, indicating that MDC1‐dependent chromatin accumulation of the MRN complex at sites of DNA breaks is not required for G2/M checkpoint activation.     

The importance of making ends meet: mutations in genes and altered expression of proteins of the MRN complex and cancer

The MRN protein complex, consisting of MRE1, RAD50 and NBS1, plays a crucial role in sensing DNA double-strand breaks (DSBs), and it is involved in cell cycle control. This makes the MRN complex an important guard of genome stability. Hypomorphic mutations in NBS1 result in the Nijmegen breakage syndrome (NBS), which is characterized by, among other things, an increased predisposition to malignancies, especially leukemia/lymphoma. Relatives of NBS patients carrying heterozygous mutations are also more prone to cancer development. This review summarizes several studies searching for associations between heterozygous mutations in NBS1, MRE11, and RAD50 and cancer and examining the levels of expression of proteins coded by these genes in tumor tissues. The results indicate that both decreased and increased expression of NBS1 may contribute to tumorigenesis, whereas overexpressed RAD50 has an anti-tumoric effect. MRE11 and RAD50 are also affected in tumors with microsatellite instability. However, the outcomes of association studies, which concerned primarily lymphomas/leukemias and breast cancer, were inconclusive. Heterozygous NBS1 mutations and molecular variants 657del5, I171V, R215W and E185Q were most commonly analyzed. Among these, an association with cancer was found most frequently for 657del5 (in leukemia/lymphoma and breast cancer) and I171V (in leukemia, breast, head and neck and colorectal cancers); however, other studies gave contradictory results. For other NBS1 as well as MRE11 and RAD50 variants, too little data were available to assess their role in cancer risk. Overall, the results suggest that heterozygous MRN complex mutations and molecular variants may contribute only to a limited fraction of tumors. This may be caused by several factors: various frequencies of the variants in specific populations, different criteria used for selection of control groups, possible effects of environmental factors, and potential interactions with variants of other low-risk genes. These issues, as well as the impact of the alterations on protein function, need to be addressed in future studies.