Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex

Studies of human Nijmegen breakage syndrome (NBS) cells have led to the proposal that the Mre11/Rad50/ NBS1 complex, which is involved in the repair of DNA double-strand breaks (DSBs), might also function in activating the DNA damage checkpoint pathways after DSBs occur. We have studied the role of the homologous budding yeast complex, Mre11/Rad50/Xrs2, in checkpoint activation in response to DSB-inducing agents. Here we show that this complex is required for phosphorylation and activation of the Rad53 and Chk1 checkpoint kinases specifically in response to DSBs. Consistent with defective Rad53 activation, we observed defective cell-cycle delays after induction of DSBs in the absence of Mre11. Furthermore, after gamma-irradiation phosphorylation of Rad9, which is an early event in checkpoint activation, is also dependent on Mre11. All three components of the Mre11/Rad50/Xrs2 complex are required for activation of Rad53, however, the Ku80, Rad51 or Rad52 proteins, which are also involved in DSB repair, are not. Thus, the integrity of the Mre11/Rad50/Xrs2 complex is specifically required for checkpoint activation after the formation of DSBs.     

Association of Mre11p with double-strand break sites during yeast meiosis

The repair of DNA double-strand breaks (DSBs) requires the activity of the Mre11/Rad50/Xrs2(Nbs1) complex. In Saccharomyces cerevisiae, this complex is required for both the initiation of meiotic recombination by Spo11p-catalyzed programmed DSBs and for break end resection, which is necessary for repair by homologous recombination. We report that Mre11p transiently associates with the chromatin of Spo11-dependent DSB regions throughout the genome. Mutant analyses show that Mre11p binding requires the function of all genes required for DSB formation, with the exception of RAD50. However, Mre11p binding does not require DSB formation itself, since Mre11p transiently associates with DSB regions in the catalysis-negative mutant spo11-Y135F. Mre11p release from chromatin is blocked in mutants that accumulate unresected DSBs. We propose that Mre11p is a component of a pre-DSB complex that assembles on the DSB sites, thus ensuring a tight coupling between DSB formation by Spo11p and the processing of break ends.     

Functional analysis of FHA and BRCT domains of NBS1 in chromatin association and DNA damage responses

Rad50/Mre11/NBS1 (R/M/N) is a multi-functional protein complex involved in DNA repair, cell cycle checkpoint activation, DNA replication and replication block-induced responses. Ionizing radiation (IR) induces the phosphorylation of NBS1 and nuclear foci formation of the complex. Although it has been suggested that the R/M/N complex is associated with DNA damage sites, we present here biochemical evidence for chromatin association of the complex. We show that the chromatin association of R/M/N is independent of IR and ataxia telangiectasia mutated (ATM). We also demonstrate that optimal chromatin association of the Rad50/Mre11/NBS1 proteins requires both the conserved forkhead-associated (FHA) and breast cancer C-terminus (BRCT) domains of NBS1. Moreover, both these domains of NBS1 are required for its phosphorylation on Ser343 but not on Ser278. Importantly, both the FHA and BRCT domains are essential for IR-induced foci (IRIF) formation of R/M/N and S phase checkpoint activation, but only the BRCT domain is needed for cell survival after IR. These data demonstrate that the FHA and BRCT domains of NBS1 are crucial for the functions of the R/M/N complex.     

PIKK-dependent phosphorylation of Mre11 induces MRN complex inactivation by disassembly from chromatin

The role of Mre11 phosphorylation in the cellular response to DNA double-strand breaks (DSBs) is not well understood. Here, we show that phosphorylation of Mre11 at SQ/TQ motifs by PIKKs (PI3 Kinase-related Kinases) induces MRN (Mre11–Rad50–Nbs1) complex dissociation from chromatin by reducing Mre11 affinity for DNA. Whereas phosphorylation of Mre11 at these residues is not required for DSB-induced ATM (Ataxia-Telangiectasia mutated) activation, abrogation of Mre11 dephosphorylation impairs ATM signaling. Our study provides a functional characterization of the DNA damage-induced Mre11 phosphorylation, and suggests that MRN inactivation participates in the down-regulation of damage signaling during checkpoint recovery following DSB repair.     

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

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

Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction

The non-homologous end-joining pathway promotes direct enzymatic rejoining of DNA double-strand breaks (DSBs) and is an important determinant of genome stability in eukaryotic cells. Although previous work has shown that this pathway requires Ku, DNA-PKcs and the DNA ligase IV/XRCC4 complex, we found that these proteins alone did not promote efficient joining of cohesive-ended DNA fragments in a cell-free assay. To identify factors that were missing from the reaction, we screened fractions from HeLa cell extracts for the ability to stimulate the joining of cohesive DNA ends in a complementation assay containing other known proteins required for DNA DSB repair. We identified a factor that restored end-joining activity to the level observed in crude nuclear extracts. Factor activity copurified with Rad50, Mre11 and NBS1, three proteins that have previously been implicated in DSB repair by genetic and cytologic evidence. Factor activity was inhibited by anti-Mre11 antibody. The reconstituted system remained fully dependent on DNL IV/XRCC4 and at least partially dependent on Ku, but the requirement for DNA-PKcs was progressively lost as other components were purified. Results support a model where DNA-PKcs acts early in the DSB repair pathway to regulate progression of the reaction, and where Mre11, Rad50 and NBS1 play a key role in aligning DNA ends in a synaptic complex immediately prior to ligation.     

Intracellular redistribution and modification of proteins of the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shock

Mre11, Rad50, and Nbs1form a tight complex which is homogeneously distributed throughout the nuclei of mammalian cells. However, after irradiation, the Mre11/Rad50/Nbs1 (M/R/N) complex rapidly migrates to sites of double strand breaks (DSBs), forming foci which remain until DSB repair is complete. Mre11 and Rad50 play direct roles in DSB repair, while Nbs1 appears to be involved in damage signaling. Hyperthermia sensitizes mammalian cells to ionizing radiation. Radiosensitization by heat shock is believed to be mediated by an inhibition of DSB repair. While the mechanism of inhibition of repair by heat shock remains to be elucidated, recent reports suggest that the M/R/N complex may be a target for inhibition of DSB repair and radiosensitization by heat. We now demonstrate that when human U-1 melanoma cells are heated at 42.5 or 45.5 degrees C, Mre11, Rad50, and Nbs1 are rapidly translocated from the nucleus to the cytoplasm. Interestingly, when cells were exposed to ionizing radiation (12 Gy of X-rays) prior to heat treatment, the extent and kinetics of translocation were increased when nuclear and cytoplasmic fractions of protein were analyzed immediately after treatment. The kinetics of the translocation and subsequent relocalization back into the nucleus when cells were incubated at 37 degrees C from 30 min to 7 h following treatment were different for each protein, which suggests that the proteins redistribute independently. However, a significant fraction of the translocated proteins exist as a triple complex in the cytoplasm. Treatment with leptomycin B (LMB) inhibits the translocation of Mre11, Rad50, and Nbs1 to the cytoplasm, leading us to speculate that the relocalization of the proteins to the cytoplasm occurs via CRM1-mediated nuclear export. In addition, while Nbs1 is rapidly phosphorylated in the nuclei of irradiated cells and is critical for a normal DNA damage response, we have found that Nbs1 is rapidly phosphorylated in the cytoplasm, but not in the nucleus, of heated irradiated cells. The phosphorylation of cytoplasmic Nbs1, which cannot be inhibited by wortmannin, appears to be a unique post-translational modification in heated, irradiated cells, and coupled with our novel observations that Mre11, Rad50, and Nbs1 translocate to the cytoplasm, lend further support for a role of the M/R/N complex in thermal radiosensitization and inhibition of DSB repair.     

Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation

High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB repair via the HR DNA repair pathway. Here we found that expression of Bcl2 results in decreased HR activity and retards the repair of DSBs induced by HZE particles (i.e. ⁵⁶iron and ²⁸silicon) by inhibiting Mre11 complex activity. Exposure of cells to ⁵⁶iron or ²⁸silicon promotes Bcl2 to interact with Mre11 via the BH1 and BH4 domains. Purified Bcl2 protein directly suppresses Mre11 complex-mediated DNA resection in vitro. Expression of Bcl2 reduces the ability of Mre11 to bind DNA following exposure of cells to HZE particles. Our findings suggest that, after cellular exposure to HZE particles, Bcl2 may inhibit Mre11 complex-mediated DNA resection leading to suppression of the HR-mediated DSB repair in surviving cells, which may potentially contribute to tumor development.     

Distinct functional domains of nibrin mediate Mre11 binding, focus formation, and nuclear localization

The inherited chromosomal instability disorder Nijmegen breakage syndrome (NBS) results from truncating mutations in the NBS1 gene, which encodes the protein nibrin. Nibrin is part of a nuclear multiprotein complex that also contains the DNA repair proteins Mre11 and Rad50. Upon irradiation, this complex redistributes within the nucleus, forming distinct foci that have been implicated as sites of DNA repair. In NBS cells, nibrin is absent and Mre11 and Rad50 are cytoplasmic. In this study, the interacting domains on nibrin and Mre11 were mapped using the yeast two-hybrid system and expression of epitope-tagged constructs in NBS fibroblasts. Deletion of the carboxy-terminal 101 amino acids of nibrin eliminated its ability to interact with Mre11 and to complement the radiation sensitivity of NBS cells. However, this truncated form of nibrin could localize to the nucleus and form radiation-inducible foci. Expression of a carboxy-terminal 354-amino-acid fragment of nibrin was sufficient to direct the nuclear localization of nibrin, as well as that of Mre11 and Rad50. Despite providing some partial complementation of the radiation-sensitive phenotype, the nibrin-Mre11-Rad50 complexes in these cells were unable to form foci. These results indicate that nibrin directs not only the nuclear localization of the nibrin-Mre11-Rad50 complexes but also radiation-induced focus formation. However, direct interaction between nibrin and Mre11 is required for normal cellular survival postirradiation. Distinct domains of nibrin are required for each of these functions, focus formation, nuclear localization, and Mre11 interaction.     

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.     

Cdk1-dependent regulation of the Mre11 complex couples DNA repair pathways to cell cycle progression

Homologous recombination (HR) and non-homologous end joining (NHEJ) are the main pathways ensuring the repair of DNA double-stranded breaks (DSBs) in eukaryotes. It has long been known that cell cycle stage is a major determinant of the type of pathway used to repair DSBs in vivo. However, the mechanistic basis for the cell cycle regulation of the DNA damage response is still unclear. Here we show that a major DSB sensor, the Mre11–Rad50–Xrs2 (MRX) complex, is regulated by cell cycle-dependent phosphorylation specifically in mitosis. This modification depends on the cyclin-dependent kinase Cdc28/Cdk1, and abrogation of Xrs2 and Mre11 phosphorylation results in a marked preference for DSB repair through NHEJ. Importantly, we show that phosphorylation of the MRX complex after DNA damage and during mitosis are regulated independently of each other by Tel1/ATM and Cdc28/Cdk1 kinases. Collectively, our results unravel an intricate network of phosphoregulatory mechanisms that act through the MRX complex to modulate DSB repair efficiency during mitosis.     

Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes.

S. cerevisiae RAD50, MRE11, and XRS2 genes are required for telomere maintenance, cell cycle checkpoint signaling, meiotic recombination, and the efficient repair of DNA double-strand breaks (DSB)s by homologous recombination and nonhomologous end-joining (NHEJ). Here, we demonstrate that the complex formed by Rad50, Mre11, and Xrs2 proteins promotes intermolecular DNA joining by DNA ligase IV (Dnl4) and its associated protein Lif1. Our results show that the Rad50/Mre11/Xrs2 complex juxtaposes linear DNA molecules via their ends to form oligomers and interacts directly with Dnl4/Lif1. We also demonstrate that Rad50/Mre11/Xrs2-mediated intermolecular DNA joining is further stimulated by Hdf1/Hdf2, the yeast homolog of the mammalian Ku70/Ku80 heterodimer. These studies reveal specific functional interplay among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, and Dnl4/Lif1 complexes in NHEJ.     

The Nijmegen breakage syndrome protein is essential for Mre11 phosphorylation upon DNA damage.

The Nijmegen breakage syndrome (NBS), a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. Repair of DNA double-strand breaks by radiation is dependent on a multifunctional complex containing Rad50, Mre11, and the NBS1 gene product, p95 (NBS protein, nibrin). The role of p95 in these repair processes is unknown. Here it is demonstrated that Mre11 is hyperphosphorylated in a cell cycle-independent manner in response to treatment of cells with genotoxic agents including gamma irradiation. This response is abrogated in two independently established NBS cell lines that have undetectable levels of the p95 protein. NBS cells are also deficient for radiation-induced nuclear foci containing Mre11, while those with Rad51 are unaffected. An analysis of the kinetic relationship between Mre11 phosphorylation and the appearance of its radiation-induced foci indicates that the former precedes the latter. Together, these data suggest that specific phosphorylation of Mre11 is induced by DNA damage, and p95 is essential in this process, perhaps by recruiting specific kinases.     

A DNA damage response pathway controlled by Tel1 and the Mre11 complex.

We define a DNA damage checkpoint pathway in S. cerevisiae governed by the ATM homolog Tel1 and the Mre11 complex. In mitotic cells, the Tel1-Mre11 complex pathway triggers Rad53 activation and its interaction with Rad9, whereas in meiosis it acts via Rad9 and the Rad53 paralog Mre4/Mek1. Activation of the Tel1-Mre11 complex pathway checkpoint functions appears to depend upon the Mre11 complex as a damage sensor and, at least in meiotic cells, to depend on unprocessed DNA double-strand breaks (DSBs). The DSB repair functions of the Mre11 complex are enhanced by the pathway, suggesting that the complex both initiates and is regulated by the Tel1-dependent DSB signal. These findings demonstrate that the diverse functions of the Mre11 complex in the cellular DNA damage response are conserved in mammals and yeast.     

The P. furiosus mre11/rad50 complex promotes 5' strand resection at a DNA double-strand break

The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3' single-stranded DNA for recombinase loading and strand exchange. In thermophilic archaea, the Mre11 and Rad50 genes cluster in an operon with genes encoding a helicase, HerA, and a 5' to 3' exonuclease, NurA, suggesting a common function. Here we show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5' strand at a DNA end under physiological conditions in vitro. The 3' single-stranded DNA generated by these enzymes can be utilized by the archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in archaea and may serve as a model for DSB processing in eukaryotes.     

Aspergillus nidulans uvsBATR and scaANBS1 genes show genetic interactions during recovery from replication stress and DNA damage

The ATM/ATR kinases and the Mre11 (Mre11-Rad50-Nbs1) protein complex are central players in the cellular DNA damage response. Here we characterize possible interactions between Aspergillus nidulans uvsB(ATR) and the Mre11 complex (scaA(NBS1)). We demonstrate that there is an epistatic relationship between uvsB(ATR), the homolog of the ATR/MEC1 gene, and scaA(NBS1), the homolog of the NBS1/XRS2 gene, for both repair and checkpoint functions and that correct ScaA(NBS1) expression during recovery from replication stress depends on uvsB(ATR). In addition, we also show that the formation of UvsC foci during recovery from replication stress is dependent on both uvsB(ATR) and scaA(NBS1) function. Furthermore, ScaA(NBS1) is also dependent on uvsB(ATR) for nuclear focus formation upon the induction of DNA double-strand breaks by phleomycin. Our results highlight the extensive genetic interactions between UvsB and the Mre11 complex that are required for S-phase progression and recovery from DNA damage.     

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.     

Damage-induced BRCA1 phosphorylation by Chk2 contributes to the timing of end resection

The BRCA1 tumor suppressor plays an important role in homologous recombination (HR)-mediated DNA double-strand-break (DSB) repair. BRCA1 is phosphorylated by Chk2 kinase upon γ-irradiation, but the role of Chk2 phosphorylation is not understood. Here, we report that abrogation of Chk2 phosphorylation on BRCA1 delays end resection and the dispersion of BRCA1 from DSBs but does not affect the assembly of Mre11/Rad50/NBS1 (MRN) and CtIP at DSBs. Moreover, we show that BRCA1 is ubiquitinated by SCF^Skp2 and that abrogation of Chk2 phosphorylation impairs its ubiquitination. Our study suggests that BRCA1 is more than a scaffold protein to assemble HR repair proteins at DSBs, but that Chk2 phosphorylation of BRCA1 also serves as a built-in clock for HR repair of DSBs. BRCA1 is known to inhibit Mre11 nuclease activity. SCF^Skp2 activity appears at late G1 and peaks at S/G2, and is known to ubiquitinate phosphodegron motifs. The removal of BRCA1 from DSBs by SCF^Skp2-mediated degradation terminates BRCA1-mediated inhibition of Mre11 nuclease activity, allowing for end resection and restricting the initiation of HR to the S/G2 phases of the cell cycle.     

Damage-induced BRCA1 phosphorylation by Chk2 contributes to the timing of end resection

The BRCA1 tumor suppressor plays an important role in homologous recombination (HR)-mediated DNA double-strand-break (DSB) repair. BRCA1 is phosphorylated by Chk2 kinase upon γ-irradiation, but the role of Chk2 phosphorylation is not understood. Here, we report that abrogation of Chk2 phosphorylation on BRCA1 delays end resection and the dispersion of BRCA1 from DSBs but does not affect the assembly of Mre11/Rad50/NBS1 (MRN) and CtIP at DSBs. Moreover, we show that BRCA1 is ubiquitinated by SCF^Skp2 and that abrogation of Chk2 phosphorylation impairs its ubiquitination. Our study suggests that BRCA1 is more than a scaffold protein to assemble HR repair proteins at DSBs, but that Chk2 phosphorylation of BRCA1 also serves as a built-in clock for HR repair of DSBs. BRCA1 is known to inhibit Mre11 nuclease activity. SCF^Skp2 activity appears at late G1 and peaks at S/G2, and is known to ubiquitinate phosphodegron motifs. The removal of BRCA1 from DSBs by SCF^Skp2-mediated degradation terminates BRCA1-mediated inhibition of Mre11 nuclease activity, allowing for end resection and restricting the initiation of HR to the S/G2 phases of the cell cycle.     

Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase).

Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs). A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1. Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4). Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes. Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains. exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination. These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ. We suggest that the 5'-3' exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures. Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways.