Poly(ADP-ribose)-binding promotes Exo1 damage recruitment and suppresses its nuclease activities

Exonuclease 1 (Exo1) has important roles in DNA metabolic transactions that are essential for genome maintenance, telomere regulation and cancer suppression. However, the mechanisms for regulating Exo1 activity in these processes remain incompletely understood. Here, we report that Exo1 activity is regulated by a direct interaction with poly(ADP-ribose) (PAR), a prominent posttranslational modification at the sites of DNA damage. This PAR-binding activity promotes the early recruitment of Exo1 to sites of DNA damage, where it is retained through an interaction with PCNA, which interacts with the C-terminus of Exo1. The effects of both PAR and PCNA on Exo1 damage association are antagonized by the 14-3-3 adaptor proteins, which interact with the central domain of Exo1. Although PAR binding inhibits both the exonuclease activity and the 5′ flap endonuclease activity of purified Exo1, the pharmacological blockade of PAR synthesis does not overtly affect DNA double-strand break end resection in a cell free Xenopus egg extract. Thus, the counteracting effects of PAR on Exo1 recruitment and enzymatic activity may enable appropriate resection of DNA ends while preventing unscheduled or improper processing of DNA breaks in cells.     

Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions

The resection of DNA double-strand breaks (DSBs) to generate ssDNA tails is a pivotal event in the cellular response to these breaks. In the two-step model of resection, primarily elucidated in yeast, initial resection by Mre11-CtIP is followed by extensive resection by two distinct pathways involving Exo1 or BLM/WRN-Dna2. However, resection pathways and their exact contributions in humans in vivo are not as clearly worked out as in yeast. Here, we examined the contribution of Exo1 to DNA end resection in humans in vivo in response to ionizing radiation (IR) and its relationship with other resection pathways (Mre11-CtIP or BLM/WRN). We find that Exo1 plays a predominant role in resection in human cells along with an alternate pathway dependent on WRN. While Mre11 and CtIP stimulate resection in human cells, they are not absolutely required for this process and Exo1 can function in resection even in the absence of Mre11-CtIP. Interestingly, the recruitment of Exo1 to DNA breaks appears to be inhibited by the NHEJ protein Ku80, and the higher level of resection that occurs upon siRNA-mediated depletion of Ku80 is dependent on Exo1. In addition, Exo1 may be regulated by 53BP1 and Brca1, and the restoration of resection in BRCA1-deficient cells upon depletion of 53BP1 is dependent on Exo1. Finally, we find that Exo1-mediated resection facilitates a transition from ATM- to ATR-mediated cell cycle checkpoint signaling. Our results identify Exo1 as a key mediator of DNA end resection and DSB repair and damage signaling decisions in human cells.     

DNA resection at chromosome breaks promotes genome stability by constraining non-allelic homologous recombination

DNA double-strand breaks impact genome stability by triggering many of the large-scale genome rearrangements associated with evolution and cancer. One of the first steps in repairing this damage is 5'→3' resection beginning at the break site. Recently, tools have become available to study the consequences of not extensively resecting double-strand breaks. Here we examine the role of Sgs1- and Exo1-dependent resection on genome stability using a non-selective assay that we previously developed using diploid yeast. We find that Saccharomyces cerevisiae lacking Sgs1 and Exo1 retains a very efficient repair process that is highly mutagenic to genome structure. Specifically, 51% of cells lacking Sgs1 and Exo1 repair a double-strand break using repetitive sequences 12-48 kb distal from the initial break site, thereby generating a genome rearrangement. These Sgs1- and Exo1-independent rearrangements depend partially upon a Rad51-mediated homologous recombination pathway. Furthermore, without resection a robust cell cycle arrest is not activated, allowing a cell with a single double-strand break to divide before repair, potentially yielding multiple progeny each with a different rearrangement. This profusion of rearranged genomes suggests that cells tolerate any dangers associated with extensive resection to inhibit mutagenic pathways such as break-distal recombination. The activation of break-distal recipient repeats and amplification of broken chromosomes when resection is limited raise the possibility that genome regions that are difficult to resect may be hotspots for rearrangements. These results may also explain why mutations in resection machinery are associated with cancer.     

Exo1 and Mre11 execute meiotic DSB end resection in the protist Tetrahymena

The resection of 5′-DNA ends at a double-strand break (DSB) is an essential step in recombinational repair, as it exposes 3′ single-stranded DNA (ssDNA) tails for interaction with a repair template. In mitosis, Exo1 and Sgs1 have a conserved function in the formation of long ssDNA tails, whereas this step in the processing of programmed meiotic DSBs is less well-characterized across model organisms. In budding yeast, which has been most intensely studied in this respect, Exo1 is a major meiotic nuclease. In addition, it exerts a nuclease-independent function later in meiosis in the conversion of DNA joint molecules into ZMM-dependent crossovers. In order to gain insight into the diverse meiotic roles of Exo1, we investigated the effect of Exo1 deletion in the ciliated protist Tetrahymena. We found that Exo1 together with Mre11, but without the help of Sgs1, promotes meiotic DSB end resection. Resection is completely eliminated only if both Mre11 and Exo1 are missing. This is consistent with the yeast model where Mre11 promotes resection in the 3′–5′ direction and Exo1 in the opposite 5′–3′ direction. However, while the endonuclease activity of Mre11 is essential to create an entry site for exonucleases and hence to start resection in budding yeast, Tetrahymena Exo1 is able to create single-stranded DNA in the absence of Mre11. Excluding a possible contribution of the Mre11 cofactor Sae2 (Com1) as an autonomous endonuclease, we conclude that there exists another unknown nuclease that initiates DSB processing in Tetrahymena. Consistent with the absence of the ZMM crossover pathway in Tetrahymena, crossover formation is independent of Exo1.     

The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1

Single-stranded DNA (ssDNA) at DNA ends is an important regulator of the DNA damage response. Resection, the generation of ssDNA, affects DNA damage checkpoint activation, DNA repair pathway choice, ssDNA-associated mutation and replication fork stability. In eukaryotes, extensive DNA resection requires the nuclease Exo1 and nuclease/helicase pair: Dna2 and Sgs1^BLM. How Exo1 and Dna2-Sgs1^BLM coordinate during resection remains poorly understood. The DNA damage checkpoint clamp (the 9-1-1 complex) has been reported to play an important role in stimulating resection but the exact mechanism remains unclear. Here we show that the human 9-1-1 complex enhances the cleavage of DNA by both DNA2 and EXO1 in vitro, showing that the resection-stimulatory role of the 9-1-1 complex is direct. We also show that in Saccharomyces cerevisiae, the 9-1-1 complex promotes both Dna2-Sgs1 and Exo1-dependent resection in response to uncapped telomeres. Our results suggest that the 9-1-1 complex facilitates resection by recruiting both Dna2-Sgs1 and Exo1 to sites of resection. This activity of the 9-1-1 complex in supporting resection is strongly inhibited by the checkpoint adaptor Rad9^53BP1. Our results provide important mechanistic insights into how DNA resection is regulated by checkpoint proteins and have implications for genome stability in eukaryotes.     

Exo1 phosphorylation status controls the hydroxyurea sensitivity of cells lacking the Pol32 subunit of DNA polymerases delta and zeta

Exo1 belongs to the Rad2 family of structure-specific nucleases and possesses 5′–3′ exonuclease activity on double-stranded DNA substrates. Exo1 interacts physically with the DNA mismatch repair (MMR) proteins Msh2 and Mlh1 and is involved in the excision of the mispaired nucleotide. Independent of its role in MMR, Exo1 contributes to long-range resection of DNA double-strand break (DSB) ends to facilitate their repair by homologous recombination (HR), and was recently identified as a component of error-free DNA damage tolerance pathways. Here, we show that Exo1 activity increases the hydroxyurea sensitivity of cells lacking Pol32, a subunit of DNA polymerases δ and ζ. Both, phospho-mimicking and dephospho-mimicking exo1 mutants act as hypermorphs, as evidenced by an increase in HU sensitivity of pol32Δ cells, suggesting that they are trapped in an active form and that phosphorylation of Exo1 at residues S372, S567, S587, S692 is necessary, but insufficient, for the accurate regulation of Exo1 activity at stalled replication forks. In contrast, neither phosphorylation status is important for Exo1's role in MMR or in the suppression of genome instability in cells lacking Sgs1 helicase. This ability of an EXO1 deletion to suppress the HU hypersensitivity of pol32Δ cells is in contrast to the negative genetic interaction between deletions of EXO1 and POL32 in MMS-treated cells as well as the role of EXO1 in DNA-damage treated rad53 and mec1 mutants.     

The Mre11 nuclease is not required for 5' to 3' resection at multiple HO-induced double-strand breaks

Current hypotheses suggest the Mre11 nuclease activity could be directly involved in double-strand break (DSB) resection in the presence of a large number of DSBs or limited to processing abnormal DNA ends. To distinguish between these possibilities, we used two methods to create large numbers of DSBs in Saccharomyces cerevisiae chromosomes, without introducing other substrates for the Mre11 nuclease. Multiple DSBs were created either by expressing the HO endonuclease in strains containing several HO cut sites embedded within randomly dispersed Ty1 elements or by phleomycin treatment. Analysis of resection by single-strand DNA formation in these systems showed no difference between strains containing MRE11 or the mre11-D56N nuclease defective allele, suggesting that the Mre11 nuclease is not involved in the extensive 5' to 3' resection of DSBs. We postulate that the ionizing radiation (IR) sensitivity of mre11 nuclease-defective mutants results from the accumulation of IR-induced DNA damage that is normally processed by the Mre11 nuclease. We also report that the processivity of 5' to 3' DSB resection and the yield of repaired products are affected by the number of DSBs in a dose-dependent manner. Finally, we show that the exonuclease Exo1 is involved in the processivity of 5' to 3' resection of an HO-induced DSB at the MAT locus.     

The Mre11-Rad50-Xrs2 Complex Is Required for Yeast DNA Postreplication Repair

Yeast DNA postreplication repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 in vivo. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass.     

The RING finger ATPase Rad5p of Saccharomyces cerevisiae contributes to DNA double-strand break repair in a ubiquitin-independent manner

Tolerance to replication-blocking DNA lesions is achieved by means of ubiquitylation of PCNA, the processivity clamp for replicative DNA polymerases, by components of the RAD6 pathway. In the yeast Saccharomyces cerevisiae the ubiquitin ligase (E3) responsible for polyubiquitylation of the clamp is the RING finger protein Rad5p. Interestingly, the RING finger, responsible for the protein's E3 activity, is embedded in a conserved DNA-dependent ATPase domain common to helicases and chromatin remodeling factors of the SWI/SNF family. Here, we demonstrate that the Rad5p ATPase domain provides the basis for a function of the protein in DNA double-strand break repair via a RAD52- and Ku-independent pathway mediated by the Mre11/Rad50/Xrs2 protein complex. This activity is distinct and separable from the contribution of the RING domain to ubiquitin conjugation to PCNA. Moreover, we show that the Rad5 protein physically associates with the single-stranded DNA regions at a processed double-strand break in vivo. Our observations suggest that Rad5p is a multifunctional protein that—by means of independent enzymatic activities inherent in its RING and ATPase domains—plays a modulating role in the coordination of repair events and replication fork progression in response to various different types of DNA lesions.     

Fission Yeast Exo1 and Rqh1-Dna2 Redundantly Contribute to Resection of Uncapped Telomeres

The uncapping of telomeres induces a DNA damage response. In Schizosaccharomyces pombe, deletion of pot1 ⁺ causes telomere uncapping and rapid telomere resection, resulting in chromosome fusion. Using the nmt-pot1-aid strain, we previously reported that Pot1 shut-off causes telomere loss and chromosome fusion in S. pombe. However, the factors responsible for the resection of uncapped telomeres remain unknown. In this study, we investigated these factors and found that concomitant deletion of rqh1 ⁺ and exo1 ⁺ alleviated the loss of telomeres following Pot1 shut-off, suggesting that Rqh1 and Exo1 are redundantly involved in the resection of uncapped telomeres. We also investigated the role of Rqh1 helicase activity and found it to be essential for the resection of uncapped telomeres. Moreover, we found that Dna2 and Exo1 function redundantly in the resection of uncapped telomeres. Taken together, these results suggest that Exo1 and Rqh1-Dna2 redundantly contribute to the resection of uncapped telomeres. Therefore, our results demonstrate that nmt-pot1-aid is an important model strain to study the role of helicases and nucleases in the resection of uncapped telomeres and to improve our understanding of DNA double-strand break repair.     

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5’-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.     

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.     

DNA resection proteins Sgs1 and Exo1 are required for G1 checkpoint activation in budding yeast

Double-strand breaks (DSBs) in budding yeast trigger activation of DNA damage checkpoints, allowing repair to occur. Although resection is necessary for initiating damage-induced cell cycle arrest in G2, no role has been assigned to it in the activation of G1 checkpoint. Here we demonstrate for the first time that the resection proteins Sgs1 and Exo1 are required for efficient G1 checkpoint activation. We find in G1 arrested cells that histone H2A phosphorylation in response to ionizing radiation is independent of Sgs1 and Exo1. In contrast, these proteins are required for damage-induced recruitment of Rfa1 to the DSB sites, phosphorylation of the Rad53 effector kinase, cell cycle arrest and RNR3 expression. Checkpoint activation in G1 requires the catalytic activity of Sgs1, suggesting that it is DNA resection mediated by Sgs1 that stimulates the damage response pathway rather than protein–protein interactions with other DDR proteins. Together, these results implicate DNA resection, which is thought to be minimal in G1, as necessary for activation of the G1 checkpoint.     

Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.     

Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping

Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.     

Functional interplay of the Mre11 nuclease and Ku in the response to replication-associated DNA damage

The Mre11 complex is a central component of the DNA damage response, with roles in damage sensing, molecular bridging, and end resection. We have previously shown that in Saccharomyces cerevisiae, Ku70 (yKu70) deficiency reduces the ionizing radiation sensitivity of mre11Δ mutants. In this study, we show that yKu70 deficiency suppressed the camptothecin (CPT) and methyl methanesulfonate (MMS) sensitivity of nuclease-deficient mre11-3 and sae2Δ mutants in an Exo1-dependent manner. CPT-induced G(2)/M arrest, γ-H2AX persistence, and chromosome breaks were elevated in mre11-3 mutants. These outcomes were reduced by yKu70 deficiency. Given that the genotoxic effects of CPT are manifest during DNA replication, these data suggest that Ku limits Exo1-dependent double-strand break (DSB) resection during DNA replication, inhibiting the initial processing steps required for homology-directed repair. We propose that Mre11 nuclease- and Sae2-dependent DNA end processing, which initiates DSB resection prevents Ku from engaging DSBs, thus promoting Exo1-dependent resection. In agreement with this idea, we show that Ku affinity for binding to short single-stranded overhangs is much lower than for blunt DNA ends. Collectively, the data define a nonhomologous end joining (NHEJ)-independent, S-phase-specific function of the Ku heterodimer.     

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.     

DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination

The repair of DNA double-strand breaks by homologous recombination commences by nucleolytic degradation of the 5′-terminated strand of the DNA break. This leads to the formation of 3′-tailed DNA, which serves as a substrate for the strand exchange protein Rad51. The nucleoprotein filament then invades homologous DNA to drive template-directed repair. In this review, I discuss mainly the mechanisms of DNA end resection in Saccharomyces cerevisiae, which includes short-range resection by Mre11-Rad50-Xrs2 and Sae2, as well as processive long-range resection by Sgs1-Dna2 or Exo1 pathways. Resection mechanisms are highly conserved between yeast and humans, and analogous machineries are found in prokaryotes as well.     

The helicase FBH1 is tightly regulated by PCNA via CRL4(Cdt2)-mediated proteolysis in human cells

During replication, DNA damage can challenge replication fork progression and cell viability. Homologous Recombination (HR) and Translesion Synthesis (TLS) pathways appear as major players involved in the resumption and completion of DNA replication. How both pathways are coordinated in human cells to maintain genome stability is unclear. Numerous helicases are involved in HR regulation. Among them, the helicase FBH1 accumulates at sites of DNA damage and potentially constrains HR via its anti-recombinase activity. However, little is known about its regulation in vivo. Here, we report a mechanism that controls the degradation of FBH1 after DNA damage. Firstly, we found that the sliding clamp Proliferating Cell Nuclear Antigen (PCNA) is critical for FBH1 recruitment to replication factories or DNA damage sites. We then showed the anti-recombinase activity of FBH1 is partially dependent on its interaction with PCNA. Intriguingly, after its re-localization, FBH1 is targeted for degradation by the Cullin-ring ligase 4-Cdt2 (CRL4^Cdt2)–PCNA pathway via a PCNA-interacting peptide (PIP) degron. Importantly, expression of non-degradable FBH1 mutant impairs the recruitment of the TLS polymerase eta to chromatin in UV-irradiated cells. Thus, we propose that after DNA damage, FBH1 might be required to restrict HR and then degraded by the Cdt2–proteasome pathway to facilitate TLS pathway.     

The MRX complex plays multiple functions in resection of Yku- and Rif2-protected DNA ends

The ends of both double-strand breaks (DSBs) and telomeres undergo tightly regulated 5' to 3' resection. Resection of DNA ends, which is specifically inhibited during the G1 cell cycle phase, requires the MRX complex, Sae2, Sgs1 and Exo1. Moreover, it is negatively regulated by the non-homologous end-joining component Yku and the telomeric protein Rif2. Here, we investigate the nuclease activities that are inhibited at DNA ends by Rif2 and Yku in G1 versus G2 by using an inducible short telomere assay. We show that, in the absence of the protective function of Rif2, resection in G1 depends primarily on MRX nuclease activity and Sae2, whereas Exo1 and Sgs1 bypass the requirement of MRX nuclease activity only if Yku is absent. In contrast, Yku-mediated inhibition is relieved in G2, where resection depends on Mre11 nuclease activity, Exo1 and, to a minor extent, Sgs1. Furthermore, Exo1 compensates for a defective MRX nuclease activity more efficiently in the absence than in the presence of Rif2, suggesting that Rif2 inhibits not only MRX but also Exo1. Notably, the presence of MRX, but not its nuclease activity, is required and sufficient to override Yku-mediated inhibition of Exo1 in G2, whereas it is required but not sufficient in G1. Finally, the integrity of MRX is also necessary to promote Exo1- and Sgs1-dependent resection, possibly by facilitating Exo1 and Sgs1 recruitment to DNA ends. Thus, resection of DNA ends that are protected by Yku and Rif2 involves multiple functions of the MRX complex that do not necessarily require its nuclease activity.