Disruption of telomere maintenance by depletion of the MRE11/RAD50/NBS1 complex in cells that use alternative lengthening of telomeres

Immortalized human cells are able to maintain their telomeres by telomerase or by a recombination-mediated DNA replication mechanism known as alternative lengthening of telomeres (ALT). We showed previously that overexpression of Sp100 protein can suppress ALT and that this was associated with sequestration of the MRE11/RAD50/NBS1 (MRN) recombination protein complex by Sp100. In the present study, we determined whether MRN proteins are required for ALT activity. ALT cells were depleted of MRN proteins by small hairpin RNA-mediated knockdown, which was maintained for up to 100 population doublings. Knockdown of NBS1 had no effect on the level of RAD50 or MRE11, but knockdown of RAD50 also depleted cells of NBS1, and knockdown of MRE11 depleted cells of all three MRN proteins. Depletion of NBS1, with or without depletion of other members of the complex, resulted in inhibition of ALT-mediated telomere maintenance, as evidenced by decreased numbers of ALT-associated promyelocytic leukemia bodies and decreased telomere length. In some clones there was an initial period of rapid shortening followed by stabilization of telomere length, whereas in others there was continuous shortening at a rate within the reported range for normal human somatic cells lacking a telomere maintenance mechanism. In contrast, depletion of NBS1 in telomerase-positive cells did not result in telomere shortening. A recent study showed that NBS1 was required for the formation of extrachromosomal telomeric circles (Compton, S. A., Choi, J. H., Cesare, A. J., Ozgur, S., and Griffith, J. D. (2007) Cancer Res. 67, 1513-1519), also a marker for ALT. We conclude that the MRN complex, and especially NBS1, is required for the ALT mechanism.     

Telomere instability in a human tumor cell line expressing NBS1 with mutations at sites phosphorylated by ATM

Nijmegen breakage syndrome (NBS) is an autosomal genetic disease demonstrating a variety of phenotypic abnormalities, including premature aging, increased cancer incidence, chromosome instability, and sensitivity to ionizing radiation. The gene involved in NBS, NBS1, is part of the MRE11/RAD50/NBS1 (MRN) complex that also includes MRE11 and RAD50, which is involved in DNA repair and cell cycle regulation in response to DNA damage. The MRN complex is also involved in telomere maintenance, as demonstrated by the shortened telomeres in NBS primary human fibroblasts and the association of NBS1 with the telomere-binding protein TRF2. To learn more about how a deficiency in telomere maintenance might contribute to chromosome instability in NBS, we have investigated the stability of telomeres in two telomerase-positive human tumor cell clones, BNmt-On and BNmt-Off, expressing an inducible NBS1(S278A/S343A) gene containing mutations at serines 278 and 343 phosphorylated by ATM. The results demonstrate an increased rate of telomere loss in both clones following expression of NBS1(S278A/S343A). The absence of detectable changes in average telomere length suggests that NBS1-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. The recombination events associated with telomere loss were found to be similar to those shown previously to result in breakage/fusion/bridge cycles, suggesting that telomere loss can contribute to chromosome instability in NBS1-deficient cells. Telomere loss showed no correlation with radiosensitivity or radioresistant DNA synthesis, demonstrating that NBS1(S278A/S343A) promotes telomere loss through a separate pathway from these other phenotypes associated with NBS.     

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 glycine-arginine domain in control of the human MRE11 DNA repair protein

Human MRE11 is a key enzyme in DNA double-strand break repair and genome stability. Human MRE11 bears a glycine-arginine-rich (GAR) motif that is conserved among multicellular eukaryotic species. We investigated how this motif influences MRE11 function. Human MRE11 alone or a complex of MRE11, RAD50, and NBS1 (MRN) was methylated in insect cells, suggesting that this modification is conserved during evolution. We demonstrate that PRMT1 interacts with MRE11 but not with the MRN complex, suggesting that MRE11 arginine methylation occurs prior to the binding of NBS1 and RAD50. Moreover, the first six methylated arginines are essential for the regulation of MRE11 DNA binding and nuclease activity. The inhibition of arginine methylation leads to a reduction in MRE11 and RAD51 focus formation on a unique double-strand break in vivo. Furthermore, the MRE11-methylated GAR domain is sufficient for its targeting to DNA damage foci and colocalization with gamma-H2AX. These studies highlight an important role for the GAR domain in regulating MRE11 function at the biochemical and cellular levels during DNA double-strand break repair.     

The role of DNA damage response proteins at telomeres--an "integrative" model

Telomeres are specialized structures at chromosome ends which play the key role in chromosomal end protection. There is increasing evidence that many DNA damage response proteins are involved in telomere maintenance. For example, cells defective in DNA double strand break repair proteins including Ku, DNA-PKcs, RAD51D and the MRN (MRE11/RAD51/NBS1) complex show loss of telomere capping function. Similarly, mouse and human cells defective in ataxia telangiectasia mutated (ATM) have defective telomeres. A total of 14 mammalian DNA damage response proteins have, so far, been implicated in telomere maintenance. Recent studies indicate that three more proteins, namely BRCA1, hRad9 and PARP1 are involved in telomere maintenance. The involvement of a wide range of DNA damage response proteins at telomeres raises an important question: do telomere maintenance mechanisms constitute an integral part of DNA damage response machinery? A model termed the "integrative" model is proposed here to argue in favour of telomere maintenance being an integral part of DNA damage response. The "integrative" model is supported by the observation that a telomeric protein, TRF2, is not confined to its local telomeric environment but it migrates to the sites of DNA breakage following exposure of cells to ionizing radiation. Furthermore, even if telomeres are maintained in a non-canonical way, as in the case of Drosophila, DNA damage response proteins are still involved in telomere maintenance suggesting integration of telomere maintenance mechanisms into the DNA damage response network.     

Rad17 recruits the MRE11‐RAD50‐NBS1 complex to regulate the cellular response to DNA double‐strand breaks

The MRE11‐RAD50‐NBS1 (MRN) complex is essential for the detection of DNA double‐strand breaks (DSBs) and initiation of DNA damage signaling. Here, we show that Rad17, a replication checkpoint protein, is required for the early recruitment of the MRN complex to the DSB site that is independent of MDC1 and contributes to ATM activation. Mechanistically, Rad17 is phosphorylated by ATM at a novel Thr622 site resulting in a direct interaction of Rad17 with NBS1, facilitating recruitment of the MRN complex and ATM to the DSB, thereby enhancing ATM signaling. Repetition of these events creates a positive feedback for Rad17‐dependent activation of MRN/ATM signaling which appears to be a requisite for the activation of MDC1‐dependent MRN complex recruitment. A point mutation of the Thr622 residue of Rad17 leads to a significant reduction in MRN/ATM signaling and homologous recombination repair, suggesting that Thr622 phosphorylation is important for regulation of the MRN/ATM signaling by Rad17. These findings suggest that Rad17 plays a critical role in the cellular response to DNA damage via regulation of the MRN/ATM pathway.     

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.     

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.     

Rescue of a telomere length defect of Nijmegen breakage syndrome cells requires NBS and telomerase catalytic subunit.

Nijmegen breakage syndrome (NBS) is a rare human disease displaying chromosome instability, radiosensitivity, cancer predisposition, immunodeficiency, and other defects [1, 2]. NBS is complexed with MRE11 and RAD50 in a DNA repair complex [3-5] and is localized to telomere ends in association with TRF proteins [6, 7]. We show that blood cells from NBS patients have shortened telomere DNA ends. Likewise, cultured NBS fibroblasts that exhibit a premature growth cessation were observed with correspondingly shortened telomeres. Introduction of the catalytic subunit of telomerase, TERT, was alone sufficient to increase the proliferative capacity of NBS fibroblasts. However, NBS, but not TERT, restores the capacity of NBS cells to survive gamma irradiation damage. Strikingly, NBS promotes telomere elongation in conjunction with TERT in NBS fibroblasts. These results suggest that NBS is a required accessory protein for telomere extension. Since NBS patients have shortened telomeres, these defects may contribute to the chromosome instability and disease associated with NBS patients.     

NBS1 prevents chromatid-type aberrations through ATM-dependent interactions with SMC1

Antoccia, A., Sakamoto, S., Matsuura, S., Tauchi, H. and Komatsu, K., NBS1 Prevents Chromatid-Type Aberrations through ATM-Dependent Interactions with SMC1. Radiat. Res. 170, 345-352 (2008).Nijmegen breakage syndrome shares several common cellular features with ataxia telangiectasia, including chromosomal instability and aberrant S- and G(2)-phase checkpoint regulation. We show here that after irradiation, NBS1 interacts physically with both BRCA1 and SMC1, a component of the cohesin complex, and that their interactions are completely abolished in AT cells. It is noted that BRCA1 is required for the interaction of NBS1 with SMC1, whereas the reverse is not the case, since BRCA1 is able to bind to NBS1 in the absence of an NBS1/SMC1 interaction as observed in MRE11- or RAD50-deficient cells. This indicates that ATM and BRCA1 are upstream of the NBS1/SMC1 interaction. Furthermore, the interaction of NBS1 with SMC1 requires both conserved domains of NBS in the N-terminus and the C-terminus, since they are indispensable for binding of NBS1 to BRCA1 and to MRE11/ATM, respectively. The interaction of NBS1 with SMC1 and the resulting phosphorylation are compromised in the clones lacking either the N- or C-terminus of NBS1, and as a consequence, chromatid-type aberrations are enhanced after irradiation. Our results reveal that ATM plays a fundamental role in promoting the radiation-induced interaction of NBS1 with SMC1 in the presence of BRCA1, leading to the maintenance of chromosomal integrity.     

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.     

Human RAD50 Deficiency in a Nijmegen Breakage Syndrome-like Disorder

The MRE11/RAD50/NBN (MRN) complex plays a key role in recognizing and signaling DNA double-strand breaks (DSBs). Hypomorphic mutations in NBN (previously known as NBS1) and MRE11A give rise to the autosomal-recessive diseases Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively. To date, no disease due to RAD50 deficiency has been described. Here, we report on a patient previously diagnosed as probably having NBS, with microcephaly, mental retardation, ‘bird-like’ face, and short stature. At variance with this diagnosis, she never had severe infections, had normal immunoglobulin levels, and did not develop lymphoid malignancy up to age 23 years. We found that she is compound heterozygous for mutations in the RAD50 gene that give rise to low levels of unstable RAD50 protein. Cells from the patient were characterized by chromosomal instability; radiosensitivity; failure to form DNA damage-induced MRN foci; and impaired radiation-induced activation of and downstream signaling through the ATM protein, which is defective in the human genetic disorder ataxia-telangiectasia. These cells were also impaired in G1/S cell-cycle-checkpoint activation and displayed radioresistant DNA synthesis and G2-phase accumulation. The defective cellular phenotype was rescued by wild-type RAD50. In conclusion, we have identified and characterized a patient with a RAD50 deficiency that results in a clinical phenotype that can be classified as an NBS-like disorder (NBSLD).     

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.     

Plk1 phosphorylation of TRF1 is essential for its binding to telomeres

In a search for Polo-like kinase 1 (Plk1) interaction proteins, we have identified TRF1 (telomeric repeat binding factor 1) as a potential Plk1 target. In this communication we report further characterization of the interaction. We show that Plk1 associates with TRF1, and Plk1 phosphorylates TRF1 at Ser-435 in vivo. Moreover, Cdk1, serving as a priming kinase, phosphorylates TRF1 to generate a docking site for Plk1 toward TRF1. In the presence of nocodazole, ectopic expression of wild type TRF1 but not TRF1 with alanine mutation in the Plk1 phosphorylation site induces apoptosis in cells containing short telomeres but not in cells containing long telomeres. Unexpectedly, down-regulation of TRF1 by RNA interference affects cell proliferation and results in obvious apoptosis in cells with short telomeres but not in cells with long telomeres. Importantly, we observe that telomeric DNA binding ability of TRF1 is cell cycle-regulated and reaches a peak during mitosis. Upon phosphorylation by Plk1 in vivo and in vitro, the ability of TRF1 to bind telomeric DNA is dramatically increased. These results demonstrate that Plk1 interacts with and phosphorylates TRF1 and suggest that Plk1-mediated phosphorylation is involved in both TRF1 overexpression-induced apoptosis and its telomeric DNA binding ability.     

Functional human telomeres are recognized as DNA damage in G2 of the cell cycle

Telomeres have to be distinguished from DNA breaks that initiate a DNA damage response. Proteins involved in the DNA damage response have previously been found at telomeres in transformed cells; however, the importance of these factors for telomere function has not been understood. Here, we show that telomeres of telomerase-negative primary cells recruit Mre11, phosphorylated NBS1, and ATM in every G2 phase of the cell cycle. This recruitment correlates with a partial release of telomeric POT1; moreover, telomeres were found to be accessible to modifying enzymes at this time in the cell cycle, suggesting that they are unprotected. Degradation of the MRN complex, as well as inhibition of ATM, led to telomere dysfunction. Consequentially, we propose that a localized DNA damage response at telomeres after replication is essential for recruiting the processing machinery that promotes formation of a chromosome end protection complex.     

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.     

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.     

Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of the MRE11/RAD50/NBS1 complex

Approximately 10% of cancers overall use alternative lengthening of telomeres (ALT) instead of telomerase to prevent telomere shortening, and ALT is especially common in astrocytomas and various types of sarcomas. The hallmarks of ALT in telomerase-negative cancer cells include a unique pattern of telomere length heterogeneity, rapid changes in individual telomere lengths, and the presence of ALT-associated promyelocytic leukemia bodies (APBs) containing telomeric DNA and proteins involved in telomere binding, DNA replication, and recombination. The ALT mechanism appears to involve recombination-mediated DNA replication, but the molecular details are largely unknown. In telomerase-null Saccharomyces cerevisiae, an analogous survivor mechanism is dependent on the RAD50 gene. We demonstrate here that overexpression of Sp100, a constituent of promyelocytic leukemia nuclear bodies, sequestered the MRE11, RAD50, and NBS1 recombination proteins away from APBs. This resulted in repression of the ALT mechanism, as evidenced by progressive telomere shortening at 121 bp per population doubling, a rate within the range found in telomerase-negative normal cells, suppression of rapid telomere length changes, and suppression of APB formation. Spontaneously generated C-terminally truncated Sp100 that did not sequester the MRE11, RAD50, and NBS1 proteins failed to inhibit ALT. These findings identify for the first time proteins that are required for the ALT mechanism.     

Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage-modified chromatin

DNA double-strand breaks (DSBs) trigger accumulation of the MRE11-RAD50-Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, whose retention on the DSB-flanking chromatin facilitates survival. Chromatin retention of MRN requires the MDC1 adaptor protein, but the mechanism behind the MRN-MDC1 interaction is unknown. We show that the NBS1 subunit of MRN interacts with the MDC1 N terminus enriched in Ser-Asp-Thr (SDT) repeats. This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1. Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1-NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells. Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs. Together, these data suggest that phosphorylation of the SDT repeats in the MDC1 N terminus functions to recruit NBS1 and, thereby, increases the local concentration of MRN at the sites of chromosomal breakage.     

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.