Recruitment of Mre11 to recombination sites during meiosis

Domain: chemistry
Published: 2026-04-07
Editorial outcome: minor_revision

Abstract
The Mre11 nuclease is part of the conserved MRX complex involved in DNA double-strand break (DSB) repair. During meiosis in budding yeast, MRX is also required for Spo11-mediated programmed DSB formation to initiate homologous recombination. Recruitment of Mre11 to meiotic DSB sites depends on Rec114-Mei4 and Mer2, proposed to organize the DSB machinery via biomolecular condensation. Here, we show that Mre11 and MRX complexes form DNA-dependent, hexanediol-sensitive condensates in vitro. In vivo, Mre11 assembles into DNA damage-dependent foci during mitosis and DSB-independent foci during meiosis. Both in vitro condensates and in vivo foci require Mre11 C-terminal intrinsically-disordered region (IDR). While dispensable for vegetative DNA repair, Mre11 IDR is essential during meiosis, where it mediates interaction with Mer2 via a short -helix and contains a SUMO-interacting motif that enhances Mre11 recruitment and DSB formation. Together, these findings provide insights into the biophysical properties of Mre11 and its role in initiating meiotic recombination.

Introduction
Cells continuously experience exogenous and endogenous stress, leading to cytotoxic lesions such as DNA double-strand breaks (DSBs)1,2. If not repaired promptly and accurately, DSBs can compromise genome stability, resulting in cellular dysfunction, tumorigenesis, or cell death3. While DSBs are generally considered detrimental for genome integrity, several instances of programmed DSBs that serve a physiological purpose are also well-documented, including V(D)J recombination during lymphocyte development, immunoglobulin class-switching, and meiotic recombination4.

During meiosis, recombination initiated by programmed DNA DSBs serves two critical functions: it ensures accurate segregation of genetic material by forming physical connections between homologous chromosomes and increases genetic diversity through allelic shuffling5,6. Meiotic DSBs are generated by the highly conserved type II topoisomerase-like protein Spo11 that acts in a consortium with several accessory factors, referred to as DSB proteins7,8.

In Saccharomyces cerevisiae, the nine known DSB proteins include Rec114-Mei4 and Mer2 (RMM). These are proposed to assemble into nucleoprotein condensates along meiotic chromosomes9 that act as sub-cellular compartments that recruit other DSB proteins to stimulate DNA cleavage by controlling Spo11 dimerization10–14. This condensation model provides a mechanism that ties DSB formation to the loop-axis organization of meiotic chromosomes15,16.

DSB formation in S. cerevisiae also depends on the Mre11-Rad50-Xrs2 (MRX) complex17–20. In addition, MRX initiates the resection of DSB ends in both meiotic and mitotically cycling cells, through the endonuclease and exonuclease activities of Mre1121–27. Further, the MRX complex has notable roles in telomere maintenance, stabilization of replication forks, and viral infection28–30.

While the roles of MRX in genome stability have been studied extensively, its role in meiotic DSB formation remains poorly understood. Here, we show that Mre11 forms dynamic condensates in vitro dependent on its C-terminal disordered tail. The Mre11 C-terminus was previously shown to be required for meiotic DSB formation31,32 and was recently found to bind Spo1133. We demonstrate that the C-terminus of Mre11 also binds directly to Mer2 and contains a SUMO-interacting motif that also facilitates its recruitment to DSB sites. Thus, our work delineates multiple mechanisms whereby the Mre11 C-terminal tail specifically promotes meiotic DSB formation.

Results
Mre11 and the MRX complex assemble DNA-dependent condensates in vitro.
During meiosis and in mitotically cycling cells exposed to DNA damage, budding yeast Mre11 forms numerous foci visible by immunofluorescence microscopy32,34,35. In vitro, Mre11-Rad50 complexes have been shown to form higher-order assemblies on DNA, mediated by Rad50 oligomerization36. However, whether Mre11 itself participates in the assembly of higher-order structures has not been established.

To investigate the biochemical properties of Mre11 and the MRX complex, we purified Alexa488-labeled Mre11 and MRX complexes with an eGFP tag at the N-terminus of Mre11 (Supplementary Figs. 1a, 2a) and asked whether they undergo condensation in vitro. In the presence of plasmid DNA, we found that Mre11 and MRX form large clusters visible by fluorescence microscopy (Fig. 1a, c). While foci were almost absent when DNA was omitted, low concentrations of DNA led to few foci of very high intensity, suggesting that the limited number of DNA molecules provide nucleation points for Mre11 to agglomerate (Supplementary Fig. 1b). Time-course analysis showed a progressive increase in foci number and intensity over time (Supplementary Fig. 1c), and protein titration revealed a steep increase in foci intensity above 200 nM Mre11 (Supplementary Fig. 1d). A similar concentration-dependent increase in foci intensity was also observed for MRX condensates (Supplementary Fig. 2b). Mre11 and MRX condensation was also promoted in the presence of crowding agents (Fig. 1e, f, and Supplementary Fig. 2b). Mre11 condensation was enhanced in the presence of divalent metal ions, likely through charge neutralization (Supplementary Fig. 1e).

To address whether the condensates are reversible, we assembled the condensates for 20 minutes and treated the reactions with DNase I. While nuclease treatment strongly reduced the numbers of Mre11 and MRX foci (Fig. 1b, d), the minority of Mre11 foci that resisted nuclease treatment tended to accumulate more protein (Supplementary Fig. 1f), although such accumulation of proteins in nuclease-resistant condensates was not observed with MRX (Fig. 1d). These observations are consistent with the idea that, following initial nucleation by DNA, condensates grow primarily through protein-protein interactions.

To further test this interpretation, we asked whether Mre11 and MRX condensates are sensitive to 1,6-hexanediol, an aliphatic alcohol that often dissolves biomolecular condensates37. Ten-minute treatment with 5% hexanediol led to a strong reduction in foci intensity (Fig. 1g, I). Similarly, brief exposure to 500 mM NaCl strongly reduced foci intensity (Fig. 1h, j). Hence, Mre11 and MRX nucleoprotein condensates are stabilized through a combination of ionic and weak hydrophobic interactions.

The C-terminal IDR of Mre11 is required for condensation.
Mre11 consists of an N-terminal phosphodiesterase domain, a capping domain followed by a Rad50 binding domain, and a C-terminal tail, predicted to be largely disordered (Fig. 2a, b)38–40. The last 15 residues form a short, conserved helix. It was previously shown that the C-terminal 49 amino acids are dispensable for mitotic DSB repair but important for Mre11 binding to meiotic DSB hotspots41 and for DSB formation31.

To identify the domain(s) required for Mre11 condensation, we purified eGFP-tagged truncations (Supplementary Fig. 3a, b). Deletion of residues 10-270 that removes most of the phosphodiesterase domain and residues 290-472 that removes the capping domain and most of the Rad50-binding domain led to reduced numbers of foci that retained high intensity (Fig. 2c). In contrast, deleting the disordered region (residues 524-677, ΔIDR) or the C-terminal 49 residues (residues 644-692, ΔC49) strongly reduced Mre11 foci intensity (Fig. 2d). Similarly, the Mre11 C-terminal extremity was also required for efficient focus formation of eGFP-tagged MRX complexes (Fig. 2e).

To understand the effects of Mre11 truncations on DNA-binding activity, we quantified binding to a pUC19 plasmid substrate by gel shift analysis (Supplementary Fig. 3c, d). All of the truncations retained significant DNA-binding activity. Similar to Mre11WT, Mre11Δ10-270 and Mre11Δ290-472 produced fast-migrating complexes at concentrations up to 25 nM protein, then formed complexes of reduced mobility at higher protein concentration, presumably indicative of higher-order assemblies (supershifts). However, these species of reduced electrophoretic mobility were absent in Mre11ΔIDR and Mre11ΔC49.

While the Mre11 IDR is required for efficient Mre11 condensation, purified eGFP-tagged Mre11IDR alone failed to form condensates and showed reduced DNA binding as compared to the wild type or Mre11ΔIDR (Supplementary Fig. 3e, f). We conclude that, while protein-DNA interactions are important for the formation of Mre11 foci in vitro, multivalent protein-protein interaction through low-complexity regions contributes to condensation.

In mitotically cycling cells, the Mre11 IDR is required for focus formation but dispensable for DNA repair.
To investigate the relationship between DNA-damage-induced Mre11 foci and DNA repair, we treated vegetative yeast cells expressing myc-tagged Mre11 with methylmethanesulfonate (MMS) and visualized focus formation by immunofluorescence microscopy. A brief 4-5 minute exposure to 5% 1,6-hexanediol readily dissolved MMS-induced foci, which instead coalesced into a few aggregates of high intensity (Fig. 3a, b).

Deletion of the IDR (mre11-ΔIDR) or C-terminal 49 residues (mre11-ΔC49) severely reduced MMS-induced focus formation relative to wild type (Fig. 3c). Despite being important for focus formation, the C-terminus of Mre11 was previously shown to be dispensable for vegetative DNA repair31. Consistently, the mre11-ΔC49 mutant was resistant to treatment with MMS or camptothecin (CPT). Similarly, mre11-ΔIDR mutants were as resistant to MMS or CPT as wild-type cells (Fig. 3d). Hence, the formation of condensate-like Mre11 foci is dispensable for DSB repair in mitotically cycling cells.

Mer2 condensates directly recruit Mre11 during meiosis.
During meiosis, Mre11 appears as discrete foci in immunofluorescence staining of prophase I chromosome spreads32,33. Analogous to mitotic cells, a brief exposure to 1,6-hexanediol disassembles these foci (Fig. 4a and Supplementary Fig. 4a). However, unlike vegetative conditions, we did not detect large Mre11 aggregates upon 1,6-hexanediol treatment during meiosis.

While Mre11 foci in mitotic cells are formed in response to DNA damage, meiotic Mre11 foci are formed independently of DSBs in cells carrying the catalytically inactive spo11-Y135F allele42 (Supplementary Fig. 4d, e). In a wild-type background, the number of Mre11 foci tended to decrease in late meiotic prophase, while foci accumulated in a spo11-Y135F mutant (Supplementary Fig. 4d). Consistent with the known meiotic function of the Mre11 C-terminus31, mre11-C49 cells produced much fewer foci in meiosis than the wild-type MRE11 cells (Fig. 4b). However, the few foci observed had an intensity similar to the wild type, suggesting that in vivo, the Mre11 C-terminus is important for the nucleation of Mre11 foci, but not their growth. In contrast, the mre11-ΔIDR strain produced fewer and much weaker foci. Approximately 40% of the cells also showed accumulation of fluorescent signal in discrete aggregates (Fig. 4b, e and Supplementary Fig. 4b, 4c). As expected, meiosis failed in both mre11-C49 and mre11-ΔIDR strains, producing only dead spores (Fig. 4c).

Meiotic foci of Mre11 depend on most other DSB proteins, including RMM41, and direct interactions have been demonstrated between Mre11, Mer243, and Spo1133. To address whether the recruitment of Mre11 during meiosis depends on RMM condensation, we quantified Mre11 foci formation in a DNA-binding defective mutant of Mer2 (mer2-KRRR) that compromises Mer2 focus formation in vitro and in vivo and abolishes DSB formation9. Similar to mer2Δ strains, the formation of Mre11 foci was strongly reduced in a mer2-KRRR mutant (Fig. 4d), despite no effect on protein levels (Supplementary Fig. 4f, g). In mer2Δ and mer2-KRRR backgrounds, Mre11 localization was analogous to the mre11-ΔIDR mutant, with few foci of low intensity and accumulation within a large aggregate (Fig. 4d, e).

To test whether Mer2 condensates can directly recruit Mre11 in vitro, we used fluorescently labelled Mre11 and Mer2, assembled their respective condensates separately on DNA substrates, and imaged the foci by microscopy. When Mer2 and Mre11 condensates were first assembled separately on a DNA substrate and then incubated together prior to imaging, we observed nearly absolute colocalization between the two proteins, supporting the hypothesis that Mer2 condensates recruit Mre11 through direct protein-protein interactions (Fig. 4f).

When these experiments were performed with the Mre11-ΔC49 truncation and wild-type Mer2, we observed strongly reduced colocalization as compared to wild-type Mre11 (Fig. 4f and Supplementary Fig. 4h), suggesting that the C-terminus of Mre11 is important for interaction with Mer2. While the C-terminus of Mre11 does not form condensates independently, in the presence of Mer2, it forms a halo-like structure around Mer2 condensates (Fig. 4g). This suggests that the interaction between Mer2 and the Mre11 C-terminus provides a nucleation site that is sufficient for further Mre11 recruitment through self-association.

The C-terminus of Mre11 binds a conserved motif of Mer2.
To delve deeper into the interaction between Mre11 and Mer2, we used AlphaFold244 to model the interaction between Mre11-C49 and a Mer2 tetramer (previously defined as the Mer2 oligomeric state9,45). AlphaFold produced a model in which the terminal -helix of Mre11 is positioned close to the N-terminal end of the Mer2 coiled coil (Fig. 5a). Interestingly, this region contains a conserved sequence motif 1 (SSM1 )46 that was previously implicated in Mre11 binding43 (Supplementary Fig. 5b). Despite the moderate confidence in the predicted interaction surface, modeling of pairs of Mer2-Mre11 homologs in different species of Saccharomycetaceae produced similar models for two out of three tested species (Supplementary Fig. 5a, Supplementary Table 6).

In the AlphaFold model, the Mre11 C-terminal helix binds anti-parallel to the Mer2 coiled coil (Fig. 5a). Mer2 residues E50, Q54, E57, and K61 (hereby referred to as EQEK) project towards the Mre11 C15 helix, and Mre11 residues L683, L686, and K690 (hereby referred to as LLK) point towards the SSM1 region of Mer2. These residues are well-conserved across Saccharomycetaceae (Supplementary Fig. 5b, d).

To test this model, we co-expressed MBP-tagged Mre11-C49 peptide and HisSUMO-tagged Mer2 in E. coli and quantified protein interactions by NiNTA pulldown. While the interaction was relatively weak, anti-MBP immunoblot analysis confirmed that Mre11-C49 directly binds Mer2 (Fig. 5b). Consistent with the AlphaFold model, alanine mutations of the Mer2-EQEK or Mre11-LLK residues strongly decreased the interaction between the two partners (Fig. 5b, c). We further validated the interaction between full-length Mer2 and Mre11 using a yeast-2-hybrid (Y2H) assay and found that mutation of the Mer2-EQEK or Mre11-LLK residues abolished the interaction (Supplementary Fig. 6a). Furthermore, truncation of the Mre11 C-terminal  helix strongly reduced co-localization between eGFP-tagged Mre11 and Alexa594-labeled Mer2 condensates in vitro (Supplementary Fig. 6b-d). Hence Mre11 binds Mer2-SSM1, and the Mre11 C-terminal 15 residues are necessary for this interaction.

In contrast to the pulldown experiments, however, the Mre11-C49 fragment was not sufficient for Mer2 binding by Y2H analysis (Supplementary Fig. 6a), suggesting that these interactions are too weak and are perhaps stabilized in the context of full-length Mre11 via dimerization of its N-terminal domain.

To establish the relevance of this interaction for Mre11 recruitment in vivo, we performed immunofluorescence analysis in strains that express the Mer2-EQEK mutant or Mre11-ΔC15 truncation. Consistent with our in vitro analysis, Mre11 focus number and intensity were reduced in both mutants, compared to the wild type (Fig. 5d). This was not due to reduced Mer2 or Mre11 expression, or reduced Mer2 chromatin association (Supplementary Fig. 6e-h).

To further verify the impact of the mutations on the interaction between Mer2 and Mre11 in vivo, we performed co-immunoprecipitation analyses on meiotic cell extracts in cells expressing V5-tagged Mer2 and myc-tagged Mre11. Mer2 was efficiently co-immunoprecipitated by Mre11, and mutation of the Mer2-EQEK motif or truncation of the Mre11 C-terminal -helix reduced this interaction (Supplementary Figs. 6i, 10e).

To address whether the interaction between Mer2 and Mre11 is important for their meiotic function, we analyzed DSB formation at the GAT1 hotspot by Southern blotting. Similar to the mre11-ΔC49 mutant, little or no DSB signal was detected in mer2-EQEK cells (Fig. 5e), while the mre11-ΔC15 mutation caused reduced and delayed DSB formation (Fig. 5f), even though the progression of meiosis was unchanged (Supplementary Fig. 6j). Consistently, mer2-EQEK mutant spores were completely inviable while the mre11-ΔC15 truncation showed 80% spore viability (Fig. 5g). Since mre11-ΔC15 does not phenocopy the mre11-ΔC49 and mer2-EQEK mutations, we conclude that the Mer2 SSM1 motif and the C-terminus of Mre11 exert other meiotic functions than the direct Mer2-Mre11 interaction identified here.

The Mre11 C-terminus contains a SUMO-interaction motif.
Several DSB proteins, including Mer2, were shown to be SUMOylated during meiosis, and SUMOylation regulates all aspects of meiotic prophase I, including DSB formation47. Notably, SUMOylation sites were mapped in the SSM1 region of Mer2 that also harbors the EQEK residues.

Mre11 has two previously identified SUMO-interacting motifs located in its phosphodiesterase domain (SIM1 and SIM2) that have been implicated in DSB repair in both mitotic and meiotic cells48. Using the GPS-SUMO tool49, we identified a third potential SIM, here called SIM3 (IIMVS), located towards the end of the Mre11 IDR (Fig. 6a and Supplementary Fig. 7a). AlphaFold prediction yielded a high-confidence model in which Mre11-SIM3, which is otherwise disordered, assumes a β-sheet structure in the presence of Smt3, as expected from a bona fide SIM50 (Supplementary Fig. 7b–7d).

To validate the Mre11-SIM3, we performed Nuclear Magnetic Resonance (NMR) spectroscopy on purified and isotopically labelled U-[13C, 15N] yeast Smt3 in the presence of a synthetic Mre11-SIM3 peptide (Fig. 6b and Supplementary Fig. 7e). As a control, we mutated residues corresponding to Mre11 I633 and M635 to alanine, predicted to contribute to the interaction with Smt3 (Supplementary Fig. 7b). NMR spectroscopy analysis indicated that the wild-type Mre11-SIM3 peptide induced strong backbone amide chemical shift to Smt3 residues I35, F36, F37, K38, I39 and R46, in contrast to the mutant peptide (Fig. 6b–6d). Residues I35-I39 form part of the second -sheet of Smt3 and R46 is located on the first -helix that are both predicted to bind the SIM3 peptide (Fig. 6e). In addition, analysis of methyl binding shifts closely agrees with the backbone amide chemical shift perturbation and further validate the AlphaFold-predicted model between Mre11-SIM3 and Smt3 (Supplementary Fig. 8).

To evaluate the affinity of Smt3 for Mre11-SIM3, we performed isothermal titration calorimetry (ITC) binding experiments between Smt3 and the Mre11-SIM3 peptides. ITC analysis confirmed that the wild-type SIM3 peptide binds Smt3 with a Kd of 20  10 µM, typical for SUMO-SIM interactions51,52. In contrast, the mutant SIM3 peptide failed to interact with Smt3 (Fig. 6f and Supplementary Fig. 9).

SIM3 contributes to Mre11 recruitment and meiotic DSB formation.
To test the role of Mre11-SIM3 in vivo, the core SIM3 motif (IIMVS) was substituted with alanines. mre11-SIM3 mutant cells showed a small reduction in meiotic Mre11 foci (Fig. 7a), which was associated with delayed DSB formation (Fig. 7b and Supplementary Fig. 10a). Combining the SIM3 mutation with truncation of the Mre11 C-terminal -helix (mre11-ΔC15+SIM3) further reduced Mre11 foci and abolished DSB formation (Fig. 7a, b), while protein levels remained unchanged (Supplementary Fig. 10b, c). Consequently, the mre11-SIM3 mutant had slightly reduced spore viability (86%), while mre11-ΔC15+SIM3 spores were completely inviable (Fig. 7c). The mre11-SIM3 mutant also showed a minor delay in meiotic progression, while progression was accelerated in the mre11-ΔC15+SIM3 mutant compared to the wild-type, likely due to the absence of DNA breaks (Supplementary Fig. 10d). We conclude that SIM3 promotes the recruitment of Mre11 to the meiotic DSB machinery.

To test the hypothesis that SIM3 contributes to the interaction between Mre11 and Mer2, we analyzed Mre11-Mer2 interaction by Y2H. While truncation of the terminal 15 amino acids of Mre11 abolished the interaction with Mer2, mutation of the SIM3 motif had no discernable impact (Supplementary Fig. 10e). We note, however, that Mer2 may not be SUMOylated in this system, which employs mitotically cycling cells. To overcome this issue, we analyzed Mre11-Mer2 interaction by co-immunoprecipitation in meiotic cells. Here, truncation of the Mre11 C-terminal 15 residues or mutation the SIM3 motif both reduced the interaction with Mer2, and combining the mutations abolished the interaction completely (Supplementary Fig. 10f), while showing no phenotype in vegetative conditions (Supplementary Fig. 10g, h). Hence, this demonstrates that the Mre11 C-terminal helix and SIM3 collaborate to promote Mre11 recruitment during meiosis. Nevertheless, the potential contribution of Mer2 SUMOylation to productive interaction with Mre11 remains unclear.

Methods
Preparation of expression vectors
Oligonucleotides (oligos) used in this study were purchased from Sigma-Aldrich. The sequences of the oligos used are listed in Supplementary Table 1. Plasmids generated in this study were verified by sequencing and are listed in Supplementary Table 2. Peptides used were ordered from GenScript or synthesized in the Ballet laboratory and are listed in Supplementary Table 3.

The expression vector for Mre1110xHis was produced by PCR amplification of the MRE11 gene from yeast genomic DNA (SK1 strain) using primers cb1351 and cb1352 and Gibson assembly into a BamHI and EcoRI digestion fragment of pFastBac1 to yield pCCB865. The sequence coding for eGFP was cloned into the BamHI site of pCCB865 to produce the expression vector for eGFPMre11, pCCB942. The expression vector for Rad50 was produced by PCR amplification of the RAD50 gene from SK1 genomic DNA using primers cb1353 and cb1354 and Gibson assembly into a BamHI and EcoRI digestion fragment of pFastBac1 to yield pCCB866. The expression vector for Xrs22xFlag was produced by PCR amplification of the XRS2 gene from SK1 genomic DNA using primers cb1355 and cb1356 and Gibson assembly into a BamHI and EcoRI digestion fragment of pFastBac1 to yield pCCB867.

Expression vectors for eGFPMre1110xHis-Δ10-270 (pry7), eGFPMre110xHis-Δ290-472 (pry5), eGFPMre110xHis-ΔIDR (pry6), eGFPMre110xHis-ΔC49 (pCCB943), and eGFPMre110xHis-ΔC15 (pry44) were amplified by inverse PCR from pCCB942 using primers pp26 and pp27, pp28 and pp29, pp24 and pp25, cb1435 and cb1458, pp73 and pp74 respectively. The amplified product was gel extracted, phosphorylated, and ligated to generate the truncations. The expression vector for eGFPMre11-IDR (pry41) was generated by Gibson assembly using a backbone amplified from pCCB942 with primers pp55 and pp61 and the Mre11-IDR sequence amplified from pCCB942 using primers pp59 and pp60.

The vector for expression of MBPMre11-C49 and HisSUMOMer2 (pCCB1040) was based on a pET-Duet1 vector with the sequence coding for MBPMre11-C49 cloned within the first position (SacI site) and the sequence coding for HisSUMOMer2 cloned at the second position (XhoI site). The EQEK mutations and LLK mutations were introduced by PCR amplification of pCCB1040 with primers cb1561 and cb1562, and pp84 and pp85, followed by phosphorylation and self-ligation to yield plasmids pry59 and pry61, respectively.

The expression vector for mScarletMre11-C49 (pry109) was generated by performing a three-fragment Gibson assembly of a pET28a backbone containing MBP followed by a TEV cleavage site amplified from pCCB785 using primers cb1486 and pp120, Mre11-C49 amplified from pCCB1040 using primers pp92 and pp136, and mScarlet amplified from pCCB785 using primers pp148 and pp149. Similarly, MBPMre11-C49 (pry99) was generated by two-fragment Gibson assembly of the fragment generated from amplification of pCCB785 using primers cb1486 and pp120 and Mre11-C49 amplified from pCCB1040 using primers pp136 and cb1559. Expression vectors for Alexa594Mer2 (pCCB750), HisSUMOMer2 coiled coil (pDD065) and eGFPMer2 (pCCB777) were previously described9. MBP expression construct was generated by Gibson assembly of a pET28a backbone and MBP amplified from pry99 using primers pp127 and pp120 and pp135 and pp132, respectively.

The pET28b expression vector for Smt3 (pCCB998) was a kind gift from Chris Lima73.

Plasmids for yeast 2-hybrid, pWL1592 (pGBDU-C1-Mer2), pWL1596 (pGAD-C1-Mre11), and pWL1565 (pGAD-C1) were generously provided by John Weir. Mutations in the coding region of Mre11 were introduced in pWL1596 via PCR mutagenesis using the primers RB70 and RB267 to generate pGAD-C1-Mre11-SIM3 (pNH1371), RB268 and RB269 to generate pGAD-C1-Mre11-ΔC15 (pNH1372), RB313 and RB308 to generate pGAD-C1-Mre11-LLK (pNH1423), RB309 and RB310 to generate pGAD-C1-Mre11-C49 (pNH1424). EQEK mutations were introduced into the coding region of Mer2 in pWL1592 using RB311 and RB312 to generate pGBDU-C1-Mer2-EQEK (pNH1422).

Expression and purification of recombinant proteins
Recombinant baculoviruses were produced by Bac-to-Bac Baculovirus Expression System (Invitrogen) following the manufacturer’s instructions. For every induction, 1 L culture containing 2 × 106 Spodoptera frugiperda (Sf9) cells/ml were infected with a Multiplicity of Infection (MOI) of 2.5 for each of the viruses. Viruses generated from pCCB865, pCCB866, and pCCB867 were used for expression of Mre1110xHis-Rad50-Xrs22xFLAG (MRX) and pCCB942, pCCB866, and pCCB867 were used for the expression of eGFPMre1110xHis-Rad50-Xrs22xFLAG (eGFPMRX). Full-length Mre1110xHis and eGFPMre1110xHis were expressed using viruses generated from pCCB856 and pCCB942, respectively. Mre11 truncations eGFPMre1110xHis-Δ10-270, eGFPMre1110xHis-Δ290-472, eGFPMre1110xHis-ΔIDR, and eGFPMre1110xHis-ΔC49 were expressed using viruses generated from pry7, pry5, pry6, and pCCB943, respectively.

Prior to harvest, Sf9 cells were allowed to infect for 62 hours at 27 ºC at 80 rpm, following which cells were pelleted at 500 rcf, washed once with 1x PBS, snap frozen in liquid nitrogen, and stored at -80ºC, or used for purification. All subsequent purification steps were carried out at 0 - 4 ºC.

The following protocol was used for the purification of His-tagged eGFPMre11 and eGFPMre11 truncations: Frozen pellets were resuspended in lysis buffer (25 mM HEPES, pH 7.5, 20 mM imidazole, 0.1 mM DTT, Roche Complete Tablet (11836170001), and 0.3 mM PMSF) and made up to a total volume of 35 ml. The samples were transferred to a beaker and osmotic lysis was performed by slowly adding 5 ml of 5 M NaCl (final 500 mM) and 10 ml of 50% (vol/vol) glycerol (final 10%) while gradually mixing with a stir bar for 30-40 mins. Lysed cells were centrifuged at 30,000 rpm for 30 mins and soluble fraction was used for affinity chromatography. 1 ml Ni-NTA resin (Thermo Scientific, 88223) was pre-equilibrated with wash buffer (25 mM HEPES, pH 7.5, 500 mM NaCl, 10% glycerol, 20 mM imidazole, 0.1 mM DTT, 0.3 mM PMSF) and batch incubated with the soluble fraction for 1 h. The resin was washed extensively in wash buffer and eluted with wash buffer containing 500 mM imidazole. Peak fractions were pooled and loaded onto a Superdex 200 Increase 10/300 GL column pre-equilibrated with SEC buffer (25 mM HEPES, 10% glycerol, 1 mM DTT, 2 mM EDTA, 300 mM NaCl). Following size-exclusion chromatography, fractions containing protein were concentrated using a 30 kDa MWCO Amicon ultra centrifugal filters (Millipore), aliquoted, snap froze in liquid nitrogen, and stored at -80°C.

For fluorescence labelling of Mre11, the Ni-NTA eluate was dialyzed several times to remove traces of imidazole. Labeling reaction was performed using Alexa Fluor 488 Protein Labeling Kit (Invitrogen, A10235) that has a succinimidyl ester moiety that reacts with primary amines. After 1 hour conjugation at room temperature, unbound fluorophore was removed by size-exclusion chromatography as described above.

Purification of recombinant His- and Flag-tagged eGFPMRX complexes and truncations were performed essentially as described74. Briefly, following osmotic lysis, soluble extract was used for sequential affinity chromatography with Ni-NTA resin (Thermo Scientific, 88223) and anti-FLAG M2 affinity gel (Sigma, A2220). Peak eluted fractions were pooled, aliquoted, and snap frozen.

For expression of recombinant mScarletMre11-C49, MBPMre11-C49, and MBP in E. coli, respective plasmids, i.e., pry109, pry99, and pry94 were transformed in BL21 cells and plated on LB plates containing kanamycin. Cells were then cultured in LB media at 37°C to an optical density (OD600) of 0.6. Expression was carried out for 20 hours at 16°C with 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for pry109 and pry99 and 3 hours at 37°C with 1 mM IPTG for pry94, following which cells were pelleted at 3000 rcf, washed once with 1x PBS, snap frozen in liquid nitrogen, and stored at -80ºC, or used for purification. Cell pellet was resuspended in lysis buffer containing 25 mM HEPES, 500 mM NaCl, 0.1 mM DTT, 0.01% NP40, 10% glycerol, 1 mM PMSF, Protease Inhibitor Cocktail (PIC, 1:100, Sigma), and 2 mM EDTA. Cells were lysed by sonication (15 W, 5 mins, 5 sec pulse) and centrifuged at 20,000 rpm for 20 mins. Soluble extract was incubated for 1 hour with 1.5 ml amylose resin (E8021L, NEB), pre-equilibrated with wash buffer (25 mM HEPES, 500 mM NaCl, 0.1 mM DTT, 0.01% NP40, 10% glycerol, 0.5 mM PMSF, Protease Inhibitor Cocktail (PIC, 1:200, Sigma), 2 mM EDTA). The column was washed extensively with wash buffer and then elution was performed in wash buffer containing 10 mM maltose. For pry109, peak fractions were pooled, the MBP-tag was cleaved with TEV protease overnight without rotation and then loaded on a Superdex 75 Increase 10/300 GL column pre-equilibrated with SEC buffer (25 mM HEPES, 300 mM NaCl, 10% glycerol, 2 mM EDTA). For pry99, peak fractions were pooled and loaded on a Superdex 200 Increase 10/300 GL column pre-equilibrated with SEC buffer (25 mM HEPES, 300 mM NaCl, 10% glycerol). For pry94, peak fractions were dialyzed overnight in dialysis buffer containing 25 mM HEPES, 300 mM NaCl, 10% glycerol. Fractions containing protein were concentrated in 10 kDa MWCO Amicon ultra centrifugal filters (Millipore), aliquoted, snap froze in liquid nitrogen, and stored at -80°C.

Expression and purification of eGFPMer2, and Alexa594Mer2 were performed as previously described9.

For expression of recombinant Smt36xHis in E. coli, pCCB998 was transformed in BL21 (DE3)pLysS cells and plated on LB plates containing kanamycin. Cells were then cultured in LB media at 37°C to an OD600 of 0.6. Expression was carried out for 3 hours at 37°C with 1 mM IPTG. Cells were centrifuged at 18°C at 4000 g for 15 mins and were directly resuspended in lysis buffer (20 mM NaPi, pH 6.5, 30 mM imidazole, 350 mM NaCl, 0.1 mM DTT, 1 mM PMSF). Cells were lysed by sonication (10 W, 4 mins, 4 sec pulse) and centrifuged at 38,000 g for 20 mins. Soluble fraction was incubated for 1 hour with 1.5 ml Ni-NTA resin, pre-equilibrated with wash buffer (20 mM NaPi, pH 6.5, 30 mM imidazole, 350 mM NaCl, 0.1 mM DTT, 0.1 mM PMSF). The column was washed extensively with wash buffer and then eluted in wash buffer containing 500 mM imidazole.

For the production of the doubly labeled U-[13C, 15N] Smt36xHis protein, the IPTG induction was carried out in minimal medium containing M9 salts (6.8 g/L Na2HPO4, 3 g/L KH2PO4, and 1 g/L NaCl), 2 mM MgSO4, 0.2 mM CaCl2, trace elements (60 mg/L FeSO4·7H2O, 12 mg/L MnCl2·4H2O, 8 mg/L CoCl2·6H2O, 7 mg/L ZnSO4·7H2O, 3 mg/L CuCl2·2H2O, 0.2 mg/L H3BO3, and 50 mg/L EDTA), BME vitamin mix (Sigma), and 1 g/L 15NH4Cl and 2 g/L [13C6]glucose (CortecNet) as the sole nitrogen and carbon sources, respectively. The purification protocol remained the same as described above.

In vitro condensation assays
Proteins were diluted to a 10× stock of their appropriate working concentrations in their respective storage buffers. Reactions were performed in a buffer containing 25 mM HEPES-HCl (pH 7.5), 2 mM DTT, 1 mg/ml BSA, 5% glycerol, 5 mM MgCl2, 5% PEG-3350, and NaCl. Considering the salt contributed by the protein dilution buffer, final concentration of NaCl in a reaction was adjusted to 120 mM. Unless specified otherwise, all reactions contained 400 nM Alexa488Mre11 or 100 nM eGFPMRX, 5.7 nM plasmid substrate, 5 % PEG-3350, and were imaged 30 minutes after assembly. A typical 20 µL binding reaction contained 2 µL protein of 10× stock of indicated concentration, 10 µL of 2× reaction buffer, and 150 ng of supercoiled pUC19 (5.7 nM). Typical reactions were assembled at 30°C for 30 mins with gentle mixing every 5 minutes, unless mentioned otherwise. 5 μL was dropped onto a microscope slide and covered with a coverslip. All images were captured on a Zeiss Axio Observer with a 100×/1.4 NA oil immersion objective except for images provided in Figure 4G which were captured on Leica Stellaris DMI 8 confocal microscope with a 63x/1.2 NA water immersion objective. Images were analyzed with ImageJ using a custom-made script9. In brief, 129.24 × 129.24-μm (2048 × 2048-pixel) images were thresholded to mean intensity of the background plus three times the standard deviation of the background. Masked foci were counted and the intensity inside the focus mask was integrated. Data points represent averages of at least 8-10 images per sample. Data were analyzed using Graphpad Prism 10.4.0.

Gel shift assays
Proteins were diluted to their appropriate working concentrations in their respective storage buffers. A typical 20 µL binding reaction was performed in a reaction buffer containing 25 mM HEPES-HCl (pH 7.5), 2 mM DTT, 1 mg/ml BSA, 10% glycerol, 5 mM EDTA, and NaCl adjusted to a final concentration of 100 mM, 1 nM pUC19 plasmid substrate, and the indicated concentration of protein. Reactions were assembled at 30°C for 30 mins and resolved in a 1% agarose (SeaKem LE Agarose, Lonza) at 60 V for 120 mins at 4°C. Gels were stained with SYBR Gold Nucleic Acid Gel Stain (S11497, Invitrogen) for 40 mins and visualized with Amersham Typhoon biomolecular imager (Cytiva).

Yeast targeting vectors and strain construction
Yeast strains are generated from Saccharomyces cerevisiae SK1 background and are listed in Supplementary Table 4.

To produce a yeast targeting vector, MRE11-myc8::URA3 was amplified using cb1424 and cb1425 from the genomic DNA of CBY375 (SKY1361) and cloned into TOPO vector by TOPO blunt cloning, generating pry2. Plasmids to produce mre11-ΔIDR (pry30), mre11-ΔC49 (pry24), and mre11-ΔC15 (pry42) mutants were generated by inverse PCR followed by self-ligation of pry2 using primers pp24 and pp25, cb1435 and pp46, and pp72 and pp73, respectively. Plasmids for the mre11-SIM (pry56) and mre11-SIM+ΔC15 (pry57) were generated similarly using primers pp3 and pp4 and templates pry2 and pry42, respectively. Genomic integration of wild-type and truncated MRE11-myc8::URA3 cassettes was performed by SpeI and NotI digestion of the corresponding plasmids and insertion in the endogenous MRE11 locus of strain CBY006 by ‘LiAc’-based transformation.

Plasmids to produce MER2 mutant strains were based on pMH002, which contains a MER2::HphMX cassette cloned into a TOPO-based vector, as described45. To generate mer2-EQEK, pMH002 was amplified by inverse PCR followed by ligation using primers cb1561 and cb1562 to yield pCCB1046. An internal V5 (iV5) tag was introduced between Mer2 residues 248 and 249 (Mer2iV5) using primers dam005 and dam006 on plasmid backbone pMH002 by inverse PCR followed by ligation to generate pDAM003. mer2iV5-EQEK was constructed by inverse PCR followed by ligation of pDAM003 using primers cb1451 and cb1562. Genomic integration of mer2-EQEK::HphMX and mer2iV5-EQEK::HphMX cassettes was performed by SpeI, NotI, and XmaI digestion of the respective plasmids and insertion into the endogenous MER2 locus of CBY006 by ‘LiAc’-based transformation. The MER2iV5::HphMX allele was constructed similarly following BamHI and SphI digestion of pDAM003.

All strains were genotyped by PCR and sequencing and opposite mating type was generated by crossing with CBY007. All other yeast strains were generated by crossing with appropriate genotypes listed in Supplementary Table 4.

Spore viability assay
For spore viability assay, a small patch of diploid strain was incubated in sporulation media (2% potassium acetate) at 30°C, 250 rpm for two days. After two days, 1 mL of sporulating culture was centrifuged and all but 200 µL of supernatant was removed. To digest yeast cells, 2 µL of concentrated sporulation culture was mixed with 100 µL 1 M sorbitol and 1 µL 10 mg/ml zymolyase and incubated at 30°C for 21 mins. 20 µL of digested cells were dropped on a YPD plate, left to dry for about 10 mins, and were micromanipulated using a tetrad dissector (MSM400, Singer Instruments). At least 20 tetrads were dissected for each assay and spore viability by assessed by calculating the number of viable spores after 2 days of incubation at 30°C.

Yeast culture and meiotic synchronization
Following standard protocols, strains were patched on YPG plates, mated and streaked on YPD plates, and selected diploid colonies grown in liquid YPD at 30°C, 250 rpm, overnight. For meiotic synchronization, diploid cultures grown overnight in YPD were transferred to YPA (1% yeast extract, 2% peptone, 1% potassium acetate) at OD600 0.2 and grown for 12-14 hours at 30°C, 250 rpm. Once the cultures reached OD600 between 1.2-1.6, cells were washed once with prewarmed sterile water and immediately transferred to sporulation medium supplemented with amino acids (320 µL amino acid complementation media for 100 mL of sporulation media (SPM)) and were kept shaking at 30°C, 250 rpm during the entire meiotic time-course. For MMS and CPT (Sigma) sensitivity assays, serial dilutions of overnight cultures were spotted on freshly prepared YPD-MMS or YPD-CPT plates containing indicated percentage of MMS or CPT, respectively. Plates were grown for two days at 30°C. For immunofluorescence of vegetatively growing cells, overnight cultures were refreshed by diluting to OD600 0.2 and grown for 3-4 hours to reach OD600 1.2-1.4 before subjecting to MMS or MMS followed by 5% 1,6-hexanediol treatment. For 5% 1,6-hexanediol treatment, cells were first converted to spheroplasts and then treated with 5% 1,6-hexanediol for 4-5 minutes. Spheroplasts were then immediately washed, lysed, and fixed using protocol described below.

Spreading and immunofluorescence of yeast nuclei spreads
Meiotic cultures were harvested 4 hours after transfer to SPM, washed with sterile cold water, and resuspended in 1 M sorbitol, 1× PBS (pH 7), 10 mM DTT, 0.5 mg/ml zymolyase 20T, and incubated for 30 mins at 30°C with gentle shaking. Spheroplasts were collected by centrifuging for 1 min at 1500 rpm and were washed gently with ice-cold 0.1 M SMES-1 M sorbitol. Spheroplasts were then centrifuged, lysed by adding ice-cold 0.1 M MES and 4% paraformaldehyde followed by vigorous finger-vortexing and immediately fixing on microscopy slides for 1 hour at room temperature. Slides were washed three times with 1 ml 0.4% PhotoFlo 200 solution (Kodak), air dried and stored at -20°C or directly used for processing.

Slides were blocked with 90% FBS, 1× PBS for 1 hour at room temperature in a humid chamber and then incubated with primary antibody (mouse mAb anti-myc, 1:200 (2276S, Cell Signaling Technology), rabbit anti-phospho H2NA-S129, 1:200 (ab15083, abcam)) diluted in 3% BSA, 1× PBS in a humid chamber for 2 hours at 37°C or overnight at 4°C. Slides were washed three times with 1× PBS in a Coplin jar, were incubated with secondary antibody (goat anti-mouse IgG Alexa Fluor™ Plus 488, 1:200 (A32723, Invitrogen), donkey anti-rabbit IgG Alexa Fluor™ 546, 1:200 (A10040, Invitrogen) diluted in 3% BSA, 1× PBS in a humid chamber at 37°C for 1 hour. Slides were washed in the dark three times for 5 mins with 1× PBS, mounted with Vectashield containing DAPI (Vector Labs). Images were captured on a Zeiss Axio Observer with a 100×/1.4 NA oil immersion objective and were analyzed in ImageJ.

Co-immunoprecipitation
Meiotic cultures were collected at 4 hours after transferring to SPM, washed once in ice-cold 1× PBS containing 1 mM PMSF, following which, they were transferred to 1.5 ml screw-cap tubes and were either snap frozen in liquid nitrogen or processed directly. Cell pellets were resuspended in 600 µl of IP buffer composed of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 % Triton ×100, 5 mM EDTA, 0.1% sodium deoxycholate, and protease inhibitors (1 mM PMSF, PIC Sigma 1:100, 1 µg/µl leupeptin, 125 U/ml benzonase, 1 µg/µl aprotinin). 900 µL of 0.5 mm zirconium beads were added to the cell pellet and cells were lyzed in a Precellys bead beater at 7200 rpm for 12 × 15 secons while chilling the tubes for 2 minutes on ice between each cycle. Cell extract was collected by poking a hole at the bottom of the tubes while placing the tubes on top of another 1.5 ml tubes and centrifuging at 800 g for 1 min at 4°C. Beads were washed with 500 µl of IP buffer and collected in the same tube. Cell lysate was spun again at 800 g for 5 min at 4°C and supernatant was transferred to 15 mL tubes. Sonication was performed three times in ice in Bioruptor Plus at intensity (M) in a 30 seconds ON / 30 seconds OFF cycle for 15 minutes each time. The lysate was centrifuged at 15.000 g at 4°C for 10 minutes and the supernatant (~ 800 µl) was transferred to new 1.5 ml tubes. 80 µL (10%) was collected as Input fraction and snap frozen. 1 µL of mouse monoclonal anti-myc primary antibody was added to each tube and tubes were rotated overnight at 5 rpm at 4°C. The following day, Dynabeads Protein G (30 µl per tube) were added to Protein LoBind tubes and washed two times with 1 mL IP buffer containing 1 mM PMSF using DynaMag2. The lysates with the antibody were transferred to the tubes containing Dynabeads and were rotated for 3-4 hours at 5 rpm at 4°C, following which, the Dynabeads were collected and washed four times with 1 ml IP buffer containing 1 mM PMSF. Finally, 60 µL IP buffer and 20 µL 4 × Lammelli buffer was added to the tubes and incubated for 5 mins at 95 °C while shaking at 1000 rpm. TCA extraction was performed on the input fractions by adding 8 µL 100 % TCA (final 10 % TCA) and incubating on ice for 30 mins. Precipitated proteins were solubilized in 40 µL 1 × Laemmli sample buffer incubated for 5 mins at 95 °C. Immunoblotting was performed using the protocol described below.

Western blotting of yeast meiotic extracts
Meiotic cultures at desired timepoints were harvested, washed in ice-cold water, and lysed in 20% trichloroacetic acid (TCA) by agitation in a bead beater (Precylles Evolution) using 0.5 mm zirconia/silica beads. Precipitated proteins were solubilized in Laemmli sample buffer and appropriate amounts of protein were separated by SDS-PAGE and analyzed by Western blotting. Western blotting was performed using standard protocol. Primary antibody used was mouse monoclonal anti-myc at 1:1000 dilution (2276S, Cell Signaling Technology), mouse monoclonal anti-V5 at 1:500 (R96025, Invitrogen), mouse monoclonal anti-PGK1 at 1:5000 (ab113687, Abcam) and secondary antibody used was goat anti-mouse IgG-HRP conjugated at 1:10,000 dilution (AP308P, Chemicon). Western blots were revealed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) in Amersham Imager 600 (Cytiva).

Southern Blotting
Meiotic DSB analysis by Southern blotting was performed as previously described75. In brief, synchronized cultures undergoing meiosis were collected at the indicated time points. After DNA isolation, 1 µg of genomic DNA was digested by PstI and separated on a 1% TBE-agarose gel. DNA was transferred to Amersham™ Hybond™-N+ nylon membranes (Cytiva) by vacuum transfer, hybridized with GAT1 probe (amplified with primers: 5′-CGCGCTTCACATAATGCTTCTGG, 5'-TTCAGATTCAACCAATCCAGGCTC) and developed by autoradiography.

Pull-down assay
Wild-type and mutant MBP-tagged Mre11-C49 and HisSUMO-tagged Mer2 were co-expressed in 50 mL of E. coli BL21 cultures and purified by affinity chromatography on Ni-NTA resin following a procedure similar to that described above. Briefly, cells were lysed by sonication and centrifuged at maximum speed for 30 mins at 4°C on a table-top centrifuge. A small fraction of the supernatant was collected as ‘Input’ and the remainder was incubated with 120 µL Ni-NTA resin, pre-equilibrated with wash buffer (25 mM HEPES pH 7.5, 20 mM imidazole, 0.1 mM DTT, 0.1 mM PMSF, 10% glycerol), for 1 hour on a rotating wheel at 4°C. The resin was washed twice with 2 ml in batch and five times with 2 ml on column before eluting with 250 µL of 500 mM imidazole in wash buffer. Input and elution fractions were separated by SDS-PAGE followed by immunoblotting with primary murine anti-MBP monoclonal antibody at 1:10,000 dilution (E8032S, NEB) and secondary goat anti-mouse IgG-HRP conjugated at 1:10,000 dilution (AP308P, Chemicon). Western blots were revealed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) in Amersham Imager 600 (Cytiva).

Nuclear magnetic resonance spectroscopy
The samples contained 0.3-0.6 mM of U-[13C,15N] Smt3 in 20 mM sodium phosphate, 20 mM NaCl (pH 6.5), 0.02% NaN3 and 10% D2O for the lock. All NMR spectra were acquired at 298 K on a Bruker Avance III HD 800 MHz spectrometer, equipped with a TCI cryoprobe. The NMR data were acquired in TopSpin 3.6 (Bruker) and processed in NMRPipe v.7.5 76 and analyzed in CCPNMR v. 2.4.2 77. Assignments of Smt3 backbone amide resonances were taken from literature78 and verified by 3D HNCACB, HN(CO)CACB, and 15N-edited NOESY-HSQC (mixing time 100 ms) spectra. Further assignments of methyl resonances were obtained from [1H, 13C] HSQC, 3D HBHA(CO)NH and (H)CCH TOCSY experiments performed on the wild-type SIM-bound Smt3 sample, which exhibited superior spectral quality compared to that of the free protein. The assigned 1H, 13C and 15N chemical shifts of the free and bound Smt3 have been deposited in the Biological Magnetic Resonance Bank (http://www.bmrb.wisc.edu/) under the accession number 53209.

The NMR binding experiments were performed by an incremental addition of wild-type or mutant SIM peptides (1.2 mM stocks in the working buffer) to 0.3 mM samples of U-[13C,15N] Smt3, with the spectral changes monitored in [1H,15N] HSQC spectra acquired at each increment and [1H, 13C] HSQC at the end of titration. The average chemical shift perturbations (Δδavg) were calculated as Δδavg = (ΔδX2/n + ΔδH2/2)0.5, where ΔδX and ΔδH are the chemical shift changes of the backbone amide nitrogens or methyl carbons (ΔδX) and protons (ΔδH) of Smt3 residues upon addition of 1.2 molar equivalents of SIM peptides, and n = 50 or 9 for the backbone amide and methyl groups, respectively.

Peptide synthesis
The peptides were synthesized by standard Fmoc-based solid phase peptide synthesis. Preloaded Fmoc-Glu-Wang resin (0.55 mmol/g) was swollen in N,N-dimethylformamide (DMF) and the Fmoc-deprotection steps consisted of shaking the resin in two consecutive steps of 5 minutes and 15 minutes, in 20% 4-methylpiperidine in DMF containing 0.1 M 1-hydroxybenzotriazole (HOBt). The coupling steps were performed with conventional Fmoc-protected amino acids (3 equiv.) (except for N-α-Fmoc-(O-3-methyl-pent-3-yl)aspartic acid), hexafluorophosphate benzotriazole tetramethyl uronium (HBTU, 3 equiv.) and N,N-diisopropylethylamine (6 equiv.). After synthesis of the full sequence on resin, the peptide was cleaved with trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (90/5/5, v/v). The products were purified by preparative reverse phase HPLC using an acetonitrile/water eluent mixture containing 0.1% TFA.

Isothermal titration calorimetry
The measurements were performed in 20 mM NaPi 100 mM NaCl pH 7.5 on Microcal ITC200 calorimeter at 25°C. The syringe contained 2 mM of peptide, while the cell held 150 µM or 300 µM of protein. Given the poor peptide solubility in aqueous buffers, the peptide solutions necessitated addition of 10% DMSO. For the ITC titrations, equal amounts of DMSO (10%) were included in both compartments to minimize the heat of dilution. Each titration consisted of a first injection of 0.4 µL, followed by 12-13 injections of 2 µL peptide into the cell, separated by intervals of 120 seconds. The first injection was discarded during the analysis of the data. The wild-type peptide titration on Smt3 was performed in duplicate. The Microcal LLC ITC200 Origin software was used to fit the data to a single binding site model.

Yeast two-hybrid
Bait and prey plasmids were co-transformed into yWL365 using the standard ‘LiAc’-based transformation and plated on SC-Leu-Ura selective media. At least four independent transformants were tested for Mer2-Mre11 interaction by spotting a dilution series on SC-Leu-Ura-His + 25 mM 3-AT (3-amino-1,2,4-triazole) and growing for 4-5 days at 30°C.

Statistical analysis and data visualization
All statistical analysis and graphing were performed using Graphpad Prism version 10.4.0. Student’s t-test was used for determination of statistical significance and P-value calculation (p ≥ 0.05, ns, not significant; **p  0.01; ***p  0.001; ****p  0.0001).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper aimed to characterize the mechanism(s) by which the Mre11 complex (MRX in budding yeast) promotes meiotic DSB formation in budding yeast. It has long been known that Mre11 is required for meiotic DSB formation by Spo11, a function that does not involve Mre11 nuclease activity and that involves the last 49 aminoacids of Mre11.\nUsing a combination of in vitro biochemical and biophysical assays with recombinant proteins, AlphaFold modelling and yeast genetics, they bring novel insight into the functions of the Mre11 complex involved.\nThe main conclusions are that Mre11, as well as MRX, can form DNA-dependent condensates in vitro, like the authors previously showed for the other DSB proteins, Mer2 and Rec114-Mei4. Mer2 condensates promote the nucleation of Mre11 condensates. They better characterize the previously described interaction between Mre11 and Mer2, using AlphaFold and mutation of the predicted interacting residues. The interaction is important for meiotic DSB formation and spore viability, although it is the only function explaining Mre11 requirement for DSB formation. Finally, they uncover a novel SUMO-interaction motif in Mre11, which, in addition to the last alpha-helix of Mre11 is required for DSB formation. These results are complementary to recent findings that showed that Mre11 also directly interacts with Spo11, to promote DSB formation.\nAltogether, although some results are still preliminary and the mechanism by which the MRX activates Spo11 is still not fully understood, this study brings novel important insight into the mechanisms by which the MRX promotes meiotic DSB formation, in particular through its interaction with Mer2. This study also reveals that meiotic DSB formation seems to be promoted, in vivo, by the formation of successive condensates, that may collaborate to concentrate Spo11 subunits and trigger its catalytic activity. Although the Mre11 complex is not required for meiotic breaks in mammals or plants, the general concept of condensates leading to meiotic DSB formation is of a broad interest.\nMajor comments:\n- It would be important to examine Mer2 foci in vivo for the mer2-EQEK mutant, to measure if it is still forming normal foci. Especially important since its phenotype seems to be different from that of mre11∆C15.\n- In the same line, it would also be important to assess co-IP in meiotic cells between Mer2 and Mre11∆C15 and between Mer2-EQEK and Mre11.\n- Figure S6A: the authors should repeat this experiment and add levels of a control protein, such as Pgk1, in order to say that mre11∆C15 levels were not changed. They do seem reduced here.\n-\nOther comments:\n- In Figure S2A: why is the band corresponding to Xrs2 more intense than the two others? This would need some explanation.\n- Figure S1C: the x axis seems to be wrong. It is rather time (minutes).\n- Page 4 line 104: should be ”Mre11 and MRX condensation…crowding agents (Figures 1E, 1F and S2B)”\n- Figure 1B and 1D: it appears clearly that for the MRX, the total foci intensity is nearly zero after DNAse treatment, whereas for Mre11, the total intensity is more or less preserved. So it does not seem that the remaining foci tend to accumulate more protein in the case of MRX. How to explain these results?\n- Figure 1J: focus number seems to be zero without treatment, because of the y axis scale. Can the authors give the actual numbers (in the text, or in the Figure legend)?\n- Page 5 line 124: “…important for meiotic DSB formation”. It could be added that this region is also required for Mre11 binding to DSB hotspot (ref 40)\n- Page 5 line 137 and Figure S3C, D and F: clarify what “Supershift” means (“complexes of reduced mobility”?)\n- Page 5 line 151 “…dissolved MMS-induced foci” and Figures 3A and B: the authors could remind that this is after 4-5 minutes 1,6-hexanediol treatment (as indicated in the methods section)\n- Page 6 line 175 and Figure 4E: the authors should show the data also for mer11∆C49\n- Figure S4F: in the absence of quantification, this experiment should be remade since there is almost no Mre11-Myc signal in the 0h time-point.\n- Page 7 and Figure 4F: for the ∆IDR and the ∆C49 it seems difficult to assess the colocalization since Mre11 foci are almost absent.\n- Page 6 line 208: “implicated in Mre11 binding”: should refer to Figure S5B.\n- Page 6 line 225: typo: should be “…Mer2 by approximately 45%”\n- Page 9 line 271: “delayed and reduced DSB formation”: from the two experiments shown in Figure 7B and Figure S10C, DSBs are delayed, but not reduced. This should be corrected.\n- Figure S10C: the right title above the Southern blot should be mre11-SIM3 and not mre11-∆SIM3", "reviewer_2": "This article builds on a previous study published in Nature in 2021, in which the authors reported that the yeast RMM protein complex assembles into nucleoprotein condensates along meiotic chromosomes, where it recruits Spo11 to stimulate DNA cleavage. In this new article, the authors characterize the role of the MRX complex in meiotic DSB formation. It was already known that the Mre11 C-terminus is required for meiotic DSB formation, and directly binds to several regions of Spo11 (as shown by pulldown and MST). Here, they show that in their in vitro conditions, Mre11 as well as the MRX complex are able to form condensates in the presence of DNA, and they identify that condensation relies on DNA binding but also on the presence of the Mre11 C-terminus that is not strongly involved in DNA binding but probably in uncharacterized protein-protein interactions. In cells, formation of meiotic foci of Mre11 depends on other DSB proteins. The authors having already shown that the RMM protein complex assembles into condensates, they tested if these condensates can recruit Mre11. They found that indeed, in cells with defective RMM condensation, the formation of Mre11 foci is strongly reduced. Moreover, they detect by yeast 2-hybrid a direct interaction between the RMM protein Mer2 and Mre11, and propose that this interaction is responsible for recruiting Mre11 in RMM condensates. Similarly, when expressing a Mre11 mutant deleted from all its C-terminal IDR region, the formation of Mre11 foci is strongly reduced. Deletion of the Mre11 C-terminus known to favor MRX condensation only decreases the number of MRX foci but not their intensities in cells. It also decreases colocalization with Mer2. The authors propose that the direct interaction between Mer2 and Mre11 mediates the nucleation of MRX condensates, whereas the propensity of MRX to form condensates is responsible for the growth of the condensates. Finally, the authors try to understand how Mre11 binds to Mer2, based on AlphaFold calculations, pull-down assays and immunofluorescence experiments. They study how much sumoylation regulates this interaction, using NMR, ITC, and immunofluorescence experiments.\nFigure 1 clearly shows that, in the tested conditions, Mre11 and MRX form condensates and that DNA favors nucleation of these condensates. In order to understand the molecular mechanisms of condensation, the authors tested the role of the different Mre11 regions in Figure 2. They first identified the different regions of Mre11. Therefore, in panel A, they present an AlphaFold Model of yeast Mre11. As the experimental 3D structure of this protein is available (PDB ref: 9BI4), it would be more reliable to present this structure. Moreover, in panel B, the authors present a prediction of the disorder as a function of the Mre11 sequence. Here again, from the analysis of the experimental structure, it is possible to identify the disordered regions of Mre11 bound to Rad50 and DNA: the cryo-EM structure of Mre11 is well-defined between residues 1 and 412 (phosphodiesterase and capping domains), and then between residues 442 and 522 (Rad50 binding domain). Such an analysis provides a precise definition (more reliable than a prediction) of the IDRs of yeast Mre11.\nIn Figure 2 panels C, D and E, the authors show that deleting most of the phosphodiesterase domain (Del 10-270) or the capping domain, the linker between the capping and Rad50 binding domains, and part of the Rad50 binding domain (Del 290-472) decreases the number of foci, whereas deleting the C-ter IDR or the C-ter 49 last residues decreases the intensity of foci (but strongly increases their number) in vitro. So, the folded domains contribute to foci nucleation, whereas the C-ter IDR contributes to the growth of the condensates. When the same experiment is performed with the whole MRX complex, deletion of the C-ter 49 last residues decreases both the intensity and the number of foci, so that it seems that the C49 region is involved in both the nucleation and the growth of the condensates. Can the authors comment on that?\nMore generally, I wonder how it could be checked that the truncated variants of Mre11 are soluble, especially in the absence of Rad50. In Figure S3B, the authors show a convincing CBB picture of these different variants that have been successfully purified. However, truncating part of the folded domains, as in Del 10-270 and Del 290-472, creates exposed hydrophobic residues that might favor Mre11 aggregation, thus modifying the condensation mechanisms.\nAlso, which interactions between Mre11 monomers could contribute to foci nucleation and growth? Does Mre11 dimerization contribute to the multivalent interactions involved in condensation ?\nAt this point, as the presence of DNA and the folded domains contribute to the nucleation of condensates, the authors characterize the DNA binding properties of their different Mre11 variants using EMSA. Here, as a non Mre11 expert, I don't understand how Mre11 binds to DNA. Indeed, in the cryoEM structure of Mre11/Rad50/dsDNA (PDB ref: 9BI4), Mre11 does not contact DNA. Moreover, yeast Mre11 is an acidic protein (IP around 5), only its C-terminal IDR is positively charged (the IP of its 49 Cter residues is particularly high: around 12). In the cryoEM structure of Mre11/Rad50/dsDNA, there are a few small positively charged patches at the surface of Mre11, however they are at the interface with Rad50. Was it previously reported that yeast Mre11 interacts with DNA? Does this happen in the activated state of Mre11? But then, is the interaction with Rad50 important for Mre11 to bind to DNA? Can the authors comment this point?\nIn Figure 4, the authors observed the Mre11 foci in meiosis. They found that the 49 Cter residues are important for foci nucleation, which was not observed in vitro. Thus, additional cellular partners account for this nucleation effect. They propose that the RMM complex is able to trigger nucleation of Mre11 foci. To test this in vitro, they produced condensates of Mer2 and Mre11, and found, by mixing these condensates, that the two proteins co-localize. This colocalization is mildly favored in the presence of the folded domains, but strongly depends on the presence of the IDR region of Mre11, and in particular its 49 Cter residues. How do the authors explain that the Mre11-C49 construct accumulates around Mer2 foci, but does not enter into these foci?\nIn Figure 5, the authors attempt to obtain a structural description of the interface between Mre11 and Mer2. They provide an AlphaFold2 model of the complex between 4 Mer2 molecules and 4 Mre11 C49 peptides. Indeed, they showed in a previous paper that Mer2 is a coiled-coil protein forming a tetramer (based on SEC-MALS and SAXS data). I found no Material and Methods section related to the AlphaFold2 calculations, and no scoring of the models (pLDDT plots; TM and ipTM scores). In Figure S5A, the heat maps suggest that the predictions are unreliable. Moreover, AlphaFold2 is known to poorly predict coiled-coil containing structures. From my point of view, if the AlphaFold2 models are poorly scored, they should not be shown. The authors can make hypotheses on the interfaces independently of the AlphaFold2 results. But showing / discussing AlphaFold2 models that are provided with low confidence scores is misleading: the reader is tempted to believe in these models, forgetting that they are based on no data. I understand that the authors found, by analyzing their models, that the motif SSM1 of Mre2 is involved in Mre11 binding, and this was previously shown elsewhere. But models obtained with poor scores do not reinforce the experimental data. As the authors can purify Mer2 and buy a synthetic peptide of Mre11 corresponding to C49, why didn't they use SEC-MALS and SAXS to characterize the Mer2-Mre11 complex, as previously reported for Mer2 alone? Is it because \"the interaction is relatively weak\" ? What do \"relatively weak\" mean ? Could the authors use ITC or BLI to measure the affinity between Mer2 and Mre11 ?\nIn order to identify the residues at the interface between Mer2 and Mre11, the authors present pulldown assays revealed by WB. Could it be added to the legend of Figure5B that the input and pulldown are revealed by WB using Ab against MBP? It is in the Material & Methods section, but it is essential for understanding the figure panel. There are a lot of not reproducible interaction data obtained by pulldown revealed by WB in the literature. To keep this data as a main figure panel, I would recommend to confirm this experiment using another experimental method. As for ex by BLI, coating a synthetic biotinylated Mre11-C49 peptide on streptavidin beads and adding increasing concentrations of HisSUMO-Mer2. Or by MST using a fluorescent Mre11-C49 peptide.\nAs sumoylation sites were mapped to the SSM1 region of Mer2, potentially involved in Mre11 binding, the authors searched for the impact of sumoylation on Mer2-Mre11 binding in cells. They detected 3 SUMO-interacting motifs in Mre11, two in the phosphodiesterase domain and one in the Cter IDR, just before the last 49 residues (633-637). They used AlphaFold2 to predict that yeast Smt3 binds to the third SIM (SIM3). Here again, no scoring is provided to judge about the robustness of the prediction. However, a careful NMR analysis (though at low salt concentration) demonstrated that the SIM3 peptide specifically binds to the SIM-binding groove of Smt3. The ITC analyses provided the Kd of the interaction. In Figure S9, the information on concentration is misleading: only one concentration is written so that it is unclear if this is the concentration of the Smt3 protein or the peptide. Could the authors add the control obtained by injecting the WT peptide into the buffer?\nIn general, as there is no proof that in cells SIM3 SUMOylation is important for meiosis, and as the impact of sumoylation of SIM3 on the interaction between Mer2 and Mre11 is still unclear, the analysis of the impact of sumoylation does not strengthen this study and could be moved to the supplementary figures.\nDiscussion:\nIn general, the discussion is based on experimental data that are still to be strengthen. When discussing about \"the Mer2 alleles previously reported to reduce Mre11 interaction that involve amino acids predicted to point inside the Mer2 tetrameric coiled coil\", why should Mer2 stay in its tetrameric state upon binding to Mre11 ?\nMinor points:\n1. Condensation: in the Mat & Met, the conditions used to study condensation include PEG (these conditions are precisely: 25 mM HEPES-HCl (pH 7.5), 2 mM DTT, 1 mg/ml BSA, 5% glycerol, 5 mM MgCl2, 5% PEG-3350, and 120 mM NaCl), whereas in the legend of Figure 1E,F, the impact of adding PEG is shown. Is there PEG in the dilution buffer only for the experiments displayed in Figure 1E,F ?\n2. NMR: in the last sentence of the Mat & Met sections, there is an n value that is cited but not present in the equation used for the CSP analysis.\n3. NMR and ITC: why is the salt concentration so different between the NMR and ITC conditions ? The ITC conditions are closer to physiological conditions than the NMR conditions. Is the salt concentration influencing the interaction ?", "reviewer_3": "# NCOMMS-25-61398-T\nRecruitment of Mre11 to recombination sites during meiosis\nIn this manuscript, Priyadarshini et al. reported that a widely studied DSB end processing factor, Mre11 of the MRX complex, forms condensate at DSB foci in both vegetative cells and during meiosis of S cerevisiae, and this involves a C-terminal IDR and the last C-terminal 15 amino acids that bind Mer2 and SUMO modification. Interestingly, while the condensate is dispensable for vegetative DSB repair, it is essential for meiosis. While some results are interesting and may offer new insights into the role of Mre11 in cerevisiae meiosis, the main conclusion lacks strong support and significant issues remain unanswered.\nMajor issues:\n1. The biggest issue is the unclearness of the true biological significance of the Mre11 condensate in DSB repair.\n• Why the condensate demonstrated different essentialities to vegetative DSB repair and meiosis? What is unclear is that if the condensates are part of the DSB foci and its formation involved the same protein-protein interaction principles (or same proteins and domains are involved), why are they essential for meiosis but not for DSB repair? What determines the different essentialities here?\n• This also raises additional concerns as to the importance of the Mre11 condensate concept in vegetative DSB repair. If it is not important, then what does it matter, or worse, who cares this condensate formation?\n• Then how to separate this condensate formation from the normal Mre11 foci at DSB repair site in vegetate cells?\n• While a DSB repair assay was performed for meiotic cells, it was absent for vegetate cells. Only a survival result was shown for vegetate cells expressing Mre11 mutants. Should also examine if these condensate deficient Mre11 mutants impact DSB repair in vegetate cells.\n• If condensate is essential for meiosis, can you rescue the lethality issue of the strains with Mre11-IDR or C49 deleted? Are there any analyses to see if the Mre11-IDR or the C49 residues alone can form foci in meiotic cells?\nWithout clear answers to these questions, the true biological significance of the Mre11 condensates at DSB sites remain uncertain.\n2. How exactly Mre11 condensates are formed, regulated, and contributed to DSB foci in vegetate and meiotic cells is unclear.\n• First, based on data presented, there are five regions/motifs of Mre11 shown to be involved: the IDR (~520-677), the C49 (residues 643-692), the C15 (residues 677-692), and the SUMO binding region (residues 633-637). The C15 binds to Mer2 and deletion of which caused similar level of foci reduction as the C49 deletion; yet, C15 is not essential for meiosis essentiality, whereas the C49 does. These results suggest the last 34 residues (643-677) of the IDR are important for meiosis. However, a comprehensive comparison of these above five fragments on in vitro condensate assembly with or without Mer2 is lacking. Also, how these fragments contribute to Mre11 foci formation in vegetate vs meiotic cells are unclear.\n• Second, there is the lack of DSB repair in vegetate vs meiotic cells expressing Mre11 mutants with deletion of just the above five fragments. Similarly, meiotic cell viability of these mutants is important to show.\n3. While the manuscript presented some interesting findings, often there was no discussion regarding the biological/pathological implications/significance of the findings.\n• Fig 3B has no discussion regarding the increase in the percentage of Mre11 aggregates by 1,6-HD treatment and the relationship of this increase to the reduction in Mre11 foci.\n• Fig 4A showed no Mre11 aggregates by 1,6-HD, which is different from vegetate cells and somehow may indicate a difference between these two states. But then, it did so when expressed in meiotic mer2-KRRR cells in Fig 4D. So, going back to the point above, what are exactly the difference between these foci and the large-in-size aggregates of Mre11? Are these reflecting real biological/pathological changes or just expression-induced nuance of Mre11 under different background? This also ties to the major issue #1.\nMinor issues:\n• Fig 1B & 1D: it stated that “While nuclease treatment strongly reduced the numbers of Mre11 and MRX foci, the minority of Mre11 foci that resisted nuclease treatment tended to accumulate more protein”. Where is the evidence that the residual condensate accumulated more protein? The selected representative images clearly did not support that. The quantitative intensity data in Fig 1B and 1D showed much reduced fluorescence intensities, which reflect the abundance of proteins in any condensate, for all foci (including the nuclease resisted ones). None of the quantitated foci after nuclease treatment retained an intensity level even close to the lowest data point prior to nuclease treatment in these two panels. This statement clearly does not hold.\n• Fig 4F was assembled with equal molar ratio of Mre11 and Mer2, then why a 4-fold ratio was used for Fig 4G (Mre11-C49:Mer2=4:1)? Will Mre11-C49 still coat Mer2 condensates if mixed at 1:1 ratio? In fact, if Mer2 works as a tetramer to recruit the MRX complex, it would be Mre11-C49:Mer2=1:4 in these in vitro assembly reactions.\n• Fig 5A only modeled the C49 of Mre11. What will happen if a larger domain of Mre11 (like 620-692 used in Fig S7A) is used? This is important as the authors proposed multiple motifs of Mre11 binding to Mer2. There are a lot of back-and-forth information regarding the domains that are involved in Mer2 binding and condensate formation of Mre11, which were not clearly and systematically evaluated.\n• Fig 7: The importance of the SIM3 motif in Mer2 binding in vivo is lacking. Why not performing similar pulldown assay as in Fig 5B for Mre11-SIM3 mutant and Mer2? Without this information, the model in Fig 7D and the conclusion in the text are premature.\n• Some of the selected representative images are not impressive to support the conclusions. For instance, Fig 1A and S1C hardly tell any foci intensity increases. Fig 2C did not show any reduction in foci number for Mre11Δ290-472. The IDR deletion Mre11 mutant had much weaker foci than the C49 deletion in Fig 4B; yet, the quantitation showed the opposite.\n• Figure legends are generally too simplified with much information missing. For instance, what was the conc of the eGFP-Mre11 protein in Fig S1B? What are conc of DNA in Fig 1A and 1C? What time point was the image taken for Fig 1A? Do Fig 2C-E contain a crowding agent? It would help the readers if all figure legends provided detailed information.\n• “…focus formation of Mer2-EQEK was diminished compared to wild-type Mre11 by approximately 45% (Figure 5D)”: Fig 5 D has nothing to do with Mer2 foci, but instead, it showed Mre11 foci under different backgrounds including in the Mer2-EQEK strain.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear Reviewers,\nWe thank the reviewers for their thoughtful and constructive comments on our\nmanuscript entitled “Recruitment of Mre11 to recombination sites during meiosis”.\nWe have addressed all reviewers’ comments in detail by performing additional\nexperiments and provide a detailed point-by-point response below (reviewer\ncomments are in black, responses are in blue).\nHere is a summary of the major and minor experiments added to the manuscript:\nMajor experiments\n1. To support our pulldown assays, we have performed yeast-2-hybrid\nexperiments between Mre11-Mer2 and their respective mutants (Mer2-EQEK\nand Mre11-LLK residues). Our new data in Supplementary Fig. 6a confirms\ninteraction between the wild-type proteins and show abolished Mre11-Mer2\ninteraction in the mutants.\n2. To address concerns regarding the strength of interaction between Mre11 and\nMer2, we performed biolayer interferometry experiments between Mer2 and the\nMre11 C-terminal 49 residues, which confirmed this interaction is indeed weak\n(Supplementary Figure 6e). This supports the idea that this interaction is further\nstabilized through multivalency and SUMO-SIM interactions.\n3. We further demonstrated by co-immunoprecipitation that Mre11 and Mer2\ninteract during meiosis and that Mer2-EQEK motif and Mre11 C-terminal helix\nare contribute to this interaction (Supplementary Figure 6j).\n4. In response to the reviewers’ concerns on the significance of SIM3 motif, we\nshow by co-immunoprecipitation that SIM3 motif and C-terminal helix of Mre11\ncooperate to promote interaction with Mer2 (Supplementary Figure 10f). This is\nconsistent with the synergistic effect that we demonstrated previously by\nimmunofluorescence, Southern blotting and spore viability analyses.\nMinor experiments\n1. We verified by immunofluorescence that the Mer2-EQEK mutant does not\ncompromise Mer2 recruitment (Supplementary Fig. 6i).\n2. We confirmed the Mre11-SIM3 and Mre11-∆C15 mutations do not affect MMS-\ninduced foci formation under vegetative conditions (Supplementary Fig. 10g).\n3. We further showed that deletion of the Mre11 C-terminal helix abolishes Mer2-\nMre11 co-localization in vitro (Supplementary Fig. 6d).\nWe thank the reviewers again for their valuable feedback on our manuscript.\nSincerely,\nCorentin Claeys Bouuaert\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis paper aimed to characterize the mechanism(s) by which the Mre11 complex\n(MRX in budding yeast) promotes meiotic DSB formation in budding yeast. It has long\nbeen known that Mre11 is required for meiotic DSB formation by Spo11, a\nfunction that does not involve Mre11 nuclease activity and that involves the last 49 aminoacids\nof Mre11.\nUsing a combination of in vitro biochemical and biophysical assays with recombinant\nproteins, AlphaFold modelling and yeast genetics, they bring novel insight into the\nfunctions of the Mre11 complex involved.\nThe main conclusions are that Mre11, as well as MRX, can form DNA-dependent\ncondensates in vitro, like the authors previously showed for the other DSB proteins,\nMer2 and Rec114-Mei4. Mer2 condensates promote the nucleation of Mre11\ncondensates. They better characterize the previously described interaction between\nMre11 and Mer2, using AlphaFold and mutation of the predicted interacting residues.\nThe interaction is important for meiotic DSB formation and spore viability, although it\nis the only function explaining Mre11 requirement for DSB formation. Finally, they\nuncover a novel SUMO-interaction motif in Mre11, which, in addition to the last alpha-\nhelix of Mre11 is required for DSB formation. These results are complementary to\nrecent findings that showed that Mre11 also directly interacts with Spo11, to promote\nDSB formation.\nAltogether, although some results are still preliminary and the mechanism by which\nthe MRX activates Spo11 is still not fully understood, this study brings novel important\ninsight into the mechanisms by which the MRX promotes meiotic DSB formation, in\nparticular through its interaction with Mer2. This study also reveals that meiotic DSB\nformation seems to be promoted, in vivo, by the formation of successive condensates,\nthat may collaborate to concentrate Spo11 subunits and trigger its catalytic activity.\nAlthough the Mre11 complex is not required for meiotic breaks in mammals or plants,\nthe general concept of condensates leading to meiotic DSB formation is of a broad\ninterest.\nMajor comments:\n- It would be important to examine Mer2 foci in vivo for the mer2-EQEK mutant, to\nmeasure if it is still forming normal foci. Especially important since its phenotype\nseems to be different from that of mre11∆C15.\nFollowing the reviewer’s suggestions, we analyzed the impact of the EQEK mutation\non Mer2 foci formation by immunofluorescence and have observed no significant\ndifference with the wild type (Supplementary Fig. 6i).\n- In the same line, it would also be important to assess co-IP in meiotic cells between\nMer2 and Mre11∆C15 and between Mer2-EQEK and Mre11.\nAs suggested by the reviewer, we have performed co-IP experiments from meiotic\nextracts (4h) between Mre11 and the Mer2-EQEK mutant and between Mer2 and the\nMre11-∆C15 mutant (Supplementary Fig. 6j). We observed a strong reduction in\ninteraction between Mre11 and Mer2-EQEK interaction and a moderate reduction\nbetween Mer2 and Mre11-∆C15, in line with the stronger meiotic phenotype (DSBs\nand spore viability) of the Mer2-EQEK mutant compared to that of the Mre11-∆C15\nmutant.\nTo further verify the impact of the mutations on Mer2-Mre11 interaction, we also\nanalyzed the Mer2-EQEK and Mer2-LLK mutants in yeast-2-hybrid experiments in\nvegetative cells, which confirmed that both mutations completely abolish the\ninteraction between these proteins in this context (Supplementary Fig. 6a). This\nreinforces the notion that multiple mechanisms collaborate to stabilize the interaction\nbetween Mer2 and Mre11 in meiotic cells.\nFinally, we demonstrate by co-IP that combining Mre11-∆C15 with the Mre11-SIM3\nmutation abolishes Mer2 interaction (Supplementary Fig. 10f), in line with a synergistic\neffect of these mutations on DSB formation.\n- Figure S6A: the authors should repeat this experiment and add levels of a control\nprotein, such as Pgk1, in order to say that mre11∆C15 levels were not changed. They\ndo seem reduced here.\nWe repeated the experiment and included a Pgk1 control. The updated data is now\nshown as Supplementary Fig. 6f.\nOther comments:\n- In Figure S2A: why is the band corresponding to Xrs2 more intense than the two\nothers? This would need some explanation.\nThe purified MRX complex contains a His-tag on Mre11 and a Flag-tag on Xrs2. The\ncomplex was purified by sequential affinity chromatography with Ni-NTA and anti-Flag\nresin, and partially dissociated during purification, yielding an excess of Xrs2. We have\nadded this clarification in the figure legend.\n- Figure S1C: the x axis seems to be wrong. It is rather time (minutes).\nFixed, thank you.\n- Page 4 line 104: should be ”Mre11 and MRX condensation…crowding agents\n(Figures 1E, 1F and S2B)”\nFixed, thank you.\n- Figure 1B and 1D: it appears clearly that for the MRX, the total foci intensity is nearly\nzero after DNAse treatment, whereas for Mre11, the total intensity is more or less\npreserved. So it does not seem that the remaining foci tend to accumulate more protein\nin the case of MRX. How to explain these results?\nWe thank the reviewer for raising this point and agree that the text did not accurately\nreflect that difference. We have now edited the text to more accurately describe the\nresult.\nThe behaviour of Mre11 and MRX complexes in these assays should not be compared\ntoo closely because both the presence of Rad50 and Xrs2 and the different tags can\nalter the condensation dynamics. In this case, we are not sure what causes this\ndifference. One possibility is that it is an indirect effect of different DNA-binding\nactivities of the complexes that may cause different protective effects on DNaseI\ntreatment. Other possibilities include that Mre11 alone has more hydrophobic surfaces\nexposed than the full complex, contributing to self-interactions. Given these\nuncertainties, we opted not to discuss these speculations and rather focus on the key\nresults, which are that (1) condensation is DNA-dependent and (2) that the Mre11 C-\nterminus contributes to self-assembly.\n- Figure 1J: focus number seems to be zero without treatment, because of the y axis\nscale. Can the authors give the actual numbers (in the text, or in the Figure legend)?\nWe have added the range of foci count to the Figure 1 legend.\n- Page 5 line 124: “…important for meiotic DSB formation”. It could be added that this\nregion is also required for Mre11 binding to DSB hotspot (ref 40)\nAdded, thank you.\n- Page 5 line 137 and Figure S3C, D and F: clarify what “Supershift” means\n(“complexes of reduced mobility”?)\nWe have included the following explanation in the legend of Supplementary Fig. 3.\n‘In panels C, D and F, a well-defined band is observed, suggestive of a stoichiometric\ncomplex (‘Complex’). With some constructs, species of lower electrophoretic mobility\nare detected at high protein concentration, suggestive of higher-order oligomeric\nspecies (‘supershift’). These oligomeric species depend on the Mre11 C-terminal IDR.’\n- Page 5 line 151 “…dissolved MMS-induced foci” and Figures 3A and B: the authors\ncould remind that this is after 4-5 minutes 1,6-hexanediol treatment (as indicated in\nthe methods section)\nDone.\n- Page 6 line 175 and Figure 4E: the authors should show the data also for mer11∆C49\nAdded, thank you.\n- Figure S4F: in the absence of quantification, this experiment should be remade since\nthere is almost no Mre11-Myc signal in the 0h time-point.\nWe have repeated the experiment, and the updated data are now included in Figure\nS4F.\n- Page 7 and Figure 4F: for the ∆IDR and the ∆C49 it seems difficult to assess the\ncolocalization since Mre11 foci are almost absent.\nWe thank the reviewer for pointing this out. To improve clarity, we have added a new\nset of images in Supplementary Fig. 4h for Mre11-WT, Mre11-∆IDR, and Mre11-∆C49\nin the presence of Mer2, with increased brightness of the eGFP channel for better\nvisualization of Mre11. This confirms that the co-localization between Mer2 and Mre11\ndepends on the C-terminal 49 residues of Mre11.\n- Page 6 line 208: “implicated in Mre11 binding”: should refer to Figure S5B.\nAdded, thank you.\n- Page 6 line 225: typo: should be “…Mer2 by approximately 45%”\nFixed, thank you.\n- Page 9 line 271: “delayed and reduced DSB formation”: from the two experiments\nshown in Figure 7B and Figure S10C, DSBs are delayed, but not reduced. This should\nbe corrected.\nDone.\n- Figure S10C: the right title above the Southern blot should be mre11-SIM3 and not\nmre11-∆SIM3\nFixed, thank you.\nReviewer #2 (Remarks to the Author):\nThis article builds on a previous study published in Nature in 2021, in which the authors\nreported that the yeast RMM protein complex assembles into nucleoprotein\ncondensates along meiotic chromosomes, where it recruits Spo11 to stimulate DNA\ncleavage. In this new article, the authors characterize the role of the MRX complex in\nmeiotic DSB formation. It was already known that the Mre11 C-terminus is required\nfor meiotic DSB formation, and directly binds to several regions of Spo11 (as shown\nby pulldown and MST). Here, they show that in their in vitro conditions, Mre11 as well\nas the MRX complex are able to form condensates in the presence of DNA, and they\nidentify that condensation relies on DNA binding but also on the presence of the Mre11\nC-terminus that is not strongly involved in DNA binding but probably in uncharacterized\nprotein-protein interactions. In cells, formation of meiotic foci of Mre11 depends on\nother DSB proteins. The authors having already shown that the RMM protein complex\nassembles into condensates, they tested if these condensates can recruit Mre11. They\nfound that indeed, in cells with defective RMM condensation, the formation of Mre11\nfoci is strongly reduced. Moreover, they detect by yeast 2-hybrid a direct interaction\nbetween the RMM protein Mer2 and Mre11, and propose that this interaction is\nresponsible for recruiting Mre11 in RMM condensates. Similarly, when expressing a\nMre11 mutant deleted from all its C-terminal IDR region, the formation of Mre11 foci\nis strongly reduced. Deletion of the Mre11 C-terminus known to favor MRX\ncondensation only decreases the number of MRX foci but not their intensities in cells.\nIt also decreases colocalization with Mer2. The authors propose that the direct\ninteraction between Mer2 and Mre11 mediates the nucleation of MRX condensates,\nwhereas the propensity of MRX to form condensates is responsible for the growth of\nthe condensates. Finally, the authors try to understand how Mre11 binds to Mer2,\nbased on AlphaFold calculations, pull-down assays and immunofluorescence\nexperiments. They study how much sumoylation regulates this interaction, using NMR,\nITC, and immunofluorescence experiments.\nFigure 1 clearly shows that, in the tested conditions, Mre11 and MRX form\ncondensates and that DNA favors nucleation of these condensates. In order to\nunderstand the molecular mechanisms of condensation, the authors tested the role of\nthe different Mre11 regions in Figure 2. They first identified the different regions of\nMre11. Therefore, in panel A, they present an AlphaFold Model of yeast Mre11. As\nthe experimental 3D structure of this protein is available (PDB ref: 9BI4), it would be\nmore reliable to present this structure. Moreover, in panel B, the authors present a\nprediction of the disorder as a function of the Mre11 sequence. Here again, from the\nanalysis of the experimental structure, it is possible to identify the disordered regions\nof Mre11 bound to Rad50 and DNA: the cryo-EM structure of Mre11 is well-defined\nbetween residues 1 and 412 (phosphodiesterase and capping domains), and then\nbetween residues 442 and 522 (Rad50 binding domain). Such an analysis provides a\nprecise definition (more reliable than a prediction) of the IDRs of yeast Mre11.\nWe thank the reviewer for the suggestion. We aligned the AlphaFold model of yeast\nMre11 provided in Figure 2A to the cryo-EM structure of dsDNA bound Mre11-Rad50\ncomplex and observed RMSD values of 0.99 Å and 1 Å for the two Mre11 molecules\nin the dimer, confirming strong agreement. Below we provide an aligned image of our\nmodel and the experimental structure to illustrate this.\n(Left) Mre11 domains are colour-coded the same as in Figure 2A. The cryo-EM\nstructure of Mre11 is in pale pink. The second Mre11, both Rad50 molecules, and the\nDNA are removed for clarity.\n(Right) Cryo-EM structure of dsDNA bound Mre11-Rad50 complex1. Both Mre11\nmolecules are in pale pink and magenta; Rad50 and DNA are in pale blue.\nThe conclusion is that all of the domains identified in Figure 2A are in agreement with\nthe cryoEM structure. We chose to show the AlphaFold model instead, because it\nshows the C-terminal IDR and C15 alpha helix, which are the major focus of the\nmanuscript. We have included a reference to the cryoEM structure to the paper though\n(line 112 in the main text and line 1006 in the figure legend of Fig. 2a).\nIn Figure 2 panels C, D and E, the authors show that deleting most of the\nphosphodiesterase domain (Del 10-270) or the capping domain, the linker between\nthe capping and Rad50 binding domains, and part of the Rad50 binding domain (Del\n290-472) decreases the number of foci, whereas deleting the C-ter IDR or the C-ter\n49 last residues decreases the intensity of foci (but strongly increases their number)\nin vitro. So, the folded domains contribute to foci nucleation, whereas the C-ter IDR\ncontributes to the growth of the condensates. When the same experiment is performed\nwith the whole MRX complex, deletion of the C-ter 49 last residues decreases both\nthe intensity and the number of foci, so that it seems that the C49 region is involved in\nboth the nucleation and the growth of the condensates. Can the authors comment on\nthat?\nIndeed, the Mre11 C-terminus can be involved in both nucleation and growth.\nFirst, as noted in response to reviewer 1 above, we think that the behaviour or Mre11\nand MRX complexes should not be compared too closely. Overall, we find that deleting\nthe Mre11 C-terminus reduces both foci number and intensity. Below a certain level of\nintensity, the foci count becomes more variable because it becomes more dependent\non an arbitrary threshold. This is evident in the quantification of panel D, for instance\nwhere the error bars in foci numbers become bigger with the IDR and C49 truncations.\nGiven this variability, we do not consider these numerical values to be very meaningful.\nSecond, foci number and intensity do not equate nucleation and growth, because both\nmetrics can be impacted by phenomena that are only indirectly related to nucleation\nand growth per se. For instance, a mutation that reduces self-interaction may lead to\nhigher foci number because the protein distributes more on DNA, but this doesn’t\nmean that nucleation itself is enhanced.\nFoci number is also a function of their rate of fusion but can be impacted by whether\nDNA sticks out of the condensates, which can act as a shield.\nMore generally, I wonder how it could be checked that the truncated variants of Mre11\nare soluble, especially in the absence of Rad50. In Figure S3B, the authors show a\nconvincing CBB picture of these different variants that have been successfully\npurified. However, truncating part of the folded domains, as in Del 10-270 and Del 290-472,\ncreates exposed hydrophobic residues that might favor Mre11 aggregation, thus\nmodifying the condensation mechanisms.\nAll the Mre11 truncations presented in this paper are purified under identical conditions\nand are analyzed by size-exclusion chromatography. None of the Mre11 truncations\neluted in the void volume, indicating that they are not aggregated. Hence, there is no\nissue with solubility of the complexes per se. We agree that the truncations can impact\ncondensation in various ways (perhaps through exposure of hydrophobic residues).\nNevertheless, this does not affect the main conclusion of the analysis, which is that\nMre11 condensation depends on its intrinsically disordered region.\nAlso, which interactions between Mre11 monomers could contribute to foci nucleation\nand growth? Does Mre11 dimerization contribute to the multivalent interactions\ninvolved in condensation ?\nWe thank the reviewer for raising this point. Indeed, we expect dimerization to\ncontribute to Mre11 condensation.\nOur data shows that the Mre11-IDR is necessary, but not sufficient for condensation\n(Figure S3E). Our interpretation is that the N-terminal domains contribute two key\ninteractions: DNA binding (that provides anchor points) and dimerization (that\ncontributes to multivalency). The IDR further contributes to DNA binding and self-\ninteractions. In the absence of the Mre11 N-terminus, these are presumably too weak\nto support condensation. However, in the presence of Mer2 condensates, Mer2 can\nact as nucleation points, allowing Mre11-C49 peptides alone to accumulate through\nself-interactions (Figure 4G).\nWe have clarified these ideas in the discussion (Lines 317-326).\nAt this point, as the presence of DNA and the folded domains contribute to the\nnucleation of condensates, the authors characterize the DNA binding properties of\ntheir different Mre11 variants using EMSA. Here, as a non Mre11 expert, I don't\nunderstand how Mre11 binds to DNA. Indeed, in the cryoEM structure of\nMre11/Rad50/dsDNA (PDB ref: 9BI4), Mre11 does not contact DNA. Moreover, yeast\nMre11 is an acidic protein (IP around 5), only its C-terminal IDR is positively charged\n(the IP of its 49 Cter residues is particularly high: around 12). In the cryoEM structure\nof Mre11/Rad50/dsDNA, there are a few small positively charged patches at the\nsurface of Mre11, however they are at the interface with Rad50. Was it previously\nreported that yeast Mre11 interacts with DNA? Does this happen in the activated state\nof Mre11? But then, is the interaction with Rad50 important for Mre11 to bind to DNA?\nCan the authors comment this point?\nWe thank the reviewer for this insightful point. Indeed, while it is known for several\nyears that yeast Mre11 directly interacts with DNA in vitro2 currently, no cryo-EM\nstructure of yeast Mre11 or the MR sub-complex in the active state is available.\nHowever, a recent study3 solved the cryo-EM structure of the dsDNA-bound MR\nsubcomplex in the catalytic state of Mre11 in E. coli. In this structure, the resting state\nshows Mre11’s nuclease site blocked by ATP-Rad50, with flexible Rad50 coiled coils.\nUpon DNA binding, the coiled coils zip into a rod, forming a clamp around dsDNA,\nwhile Mre11 moves to the side, binds the DNA end, and assembles a nuclease\nchannel. Based on this, Nicolas, et al.4 proposed an AlphaFold2 model of the DNA-\nbound catalytic state of yeast Mre11-Rad50, which resembles the E. coli structure, and\nprovide evidence that this configuration depends on Sae2. While in vitro EMSA\nexperiments show that Rad50 is not required for binding of Mre11 to DNA, Mre11-\nRad50 oligomerization is required for proper DNA-binding behaviour and Mre11\nendonuclease activity5.\nIn Figure 4, the authors observed the Mre11 foci in meiosis. They found that the 49\nCter residues are important for foci nucleation, which was not observed in vitro. Thus,\nadditional cellular partners account for this nucleation effect. They propose that the\nRMM complex is able to trigger nucleation of Mre11 foci. To test this in vitro, they\nproduced condensates of Mer2 and Mre11, and found, by mixing these condensates,\nthat the two proteins co-localize. This colocalization is mildly favored in the presence\nof the folded domains, but strongly depends on the presence of the IDR region of\nMre11, and in particular its 49 Cter residues. How do the authors explain that the\nMre11-C49 construct accumulates around Mer2 foci, but does not enter into these\nfoci?\nOur interpretation is that the full-length Mre11 protein enters Mer2 foci because it\nproduces networks through protein-DNA interactions, Mre11 dimerization domain,\nplus Mre11-Mre11 interactions and Mre11-Mer2 interactions. The C49 residues lack a\nkey DNA-binding and dimerization domain and seem to maintain only two properties:\ndirect interaction with Mer2 and self-interaction between C49 peptides. Our\nunderstanding is that binding to Mer2 allows Mre11-C49 to locally reach a critical\nconcentration that allows Mre11 accumulation through self-interaction. Presumably,\nMre11-C49 does not enter Mer2 foci because their homotypic interactions are higher\nthan heterotypic interactions with Mer2.\nThe above explanation is added to lines 317-326 of Discussion.\nIn Figure 5, the authors attempt to obtain a structural description of the interface\nbetween Mre11 and Mer2. They provide an AlphaFold2 model of the complex between\nthat Mer2 is a coiled-coil protein forming a tetramer (based on SEC-MALS and SAXS\ndata). I found no Material and Methods section related to the AlphaFold2 calculations,\nand no scoring of the models (pLDDT plots; TM and ipTM scores). In Figure S5A, the\nheat maps suggest that the predictions are unreliable. Moreover, AlphaFold2 is known\nto poorly predict coiled-coil containing structures. From my point of view, if the\nAlphaFold2 models are poorly scored, they should not be shown. The authors can\nmake hypotheses on the interfaces independently of the AlphaFold2 results. But\nshowing / discussing AlphaFold2 models that are provided with low confidence scores\nis misleading: the reader is tempted to believe in these models, forgetting that they are\nbased on no data. I understand that the authors found, by analyzing their models,\nthat the motif SSM1 of Mre2 is involved in Mre11 binding, and this was previously\nshown elsewhere. But models obtained with poor scores do not reinforce the experimental\ndata. As the authors can purify Mer2 and buy a synthetic peptide of Mre11\ncorresponding to C49, why didn't they use SEC-MALS and SAXS to characterize the\nMer2-Mre11 complex, as previously reported for Mer2 alone? Is it because \"the\ninteraction is relatively weak\" ? What do \"relatively weak\" mean ? Could the authors\nuse ITC or BLI to measure the affinity between Mer2 and Mre11 ?\nWe thank the reviewer for raising the concern regarding the presented AlphaFold\nmodels. We have included iPTM scores for the models in Table S6. While we agree\nwith the reviewer that the AlphaFold models have low confidence scores, our intention\nbehind adding the models was to show a hypothesis-driving illustration to highlight the\npotential spatial arrangement of Mre11-C49 in presence of tetrameric Mer2. Indeed,\nthe AlphaFold models alone do not form the basis of a mechanistic conclusion.\nFollowing the reviewer’s suggestions, we performed BLI experiments of Mre11-C49\nand Mer2 coiled-coil and found a K value of 1.16 mM, indicative of a weak interaction\nD\nbetween both the proteins. This data is added to Supplementary Figure 6e.\nNote that this weak interaction does not undermine the biological importance of this\ninteraction. First, in the context of the full-length protein, the interaction will be\nreinforced by the dimeric stoichiometry of Mre11. Second, we show that during\nmeiosis, this interaction works together with a SUMO-SIM interaction to allow Mre11\nrecruitment.\nWe also attempted ITC experiments between Mre11-C49 with Mer2 coiled-coil.\nTitration of Mre11-C49 into Mer2 produced endothermic peaks that also appeared\nwhen the peptide was titrated into buffer, rendering the results inconclusive (see\nbelow).\nThese data indicate that the interaction is indeed weak, as we had deduced from the\npulldown analyses. Biophysical characterizations via SEC-MALS and SAXS are\ntherefore not feasible.\nIn order to identify the residues at the interface between Mer2 and Mre11, the authors\npresent pulldown assays revealed by WB. Could it be added to the legend of Figure5B\nthat the input and pulldown are revealed by WB using Ab against MBP? It is in the\nMaterial & Methods section, but it is essential for understanding the figure panel.\nAdded to the legend, thank you.\nThere are a lot of not reproducible interaction data obtained by pulldown revealed by\nWB in the literature. To keep this data as a main figure panel, I would recommend to\nconfirm this experiment using another experimental method. As for ex by BLI,\ncoating a synthetic biotinylated Mre11-C49 peptide on streptavidin beads and adding\nincreasing concentrations of HisSUMO-Mer2. Or by MST using a fluorescent Mre11-\nC49 peptide.\nWe thank the reviewer for the suggestion. To complement our pulldown experiments,\nwe performed the following additional experiments:\n BLI: Mre11-C49 and Mer2 coiled-coil interaction show a K of 1.16 mM, indicative\nD\nof a weak interaction (Supplementary Fig. 6e).\n Yeast-2-hybrid: Interaction between Mer2 and Mre11-LLK and between Mre11 and\nMer2-EQEK was abolished (Supplementary Fig. 6a).\n Co-immunoprecipitation in meiotic cells: We observed interaction between Mre11\nand Mer2, while Mre11-ΔC15 and Mer2 and Mer2-EQEK and Mre11 showed\nreduced interaction (Supplementary Figs. 6j, 10f).\nAs sumoylation sites were mapped to the SSM1 region of Mer2, potentially involved\nin Mre11 binding, the authors searched for the impact of sumoylation on Mer2-Mre11\nbinding in cells. They detected 3 SUMO-interacting motifs in Mre11, two in the\nphosphodiesterase domain and one in the Cter IDR, just before the last 49 residues\n(633-637). They used AlphaFold2 to predict that yeast Smt3 binds to the third SIM\n(SIM3). Here again, no scoring is provided to judge about the robustness of the\nprediction. However, a careful NMR analysis (though at low salt concentration) demonstrated\nthat the SIM3 peptide specifically binds to the SIM-binding groove of\nSmt3. The ITC analyses provided the Kd of the interaction. In Figure S9, the\ninformation on concentration is misleading: only one concentration is written so that it\nis unclear if this is the concentration of the Smt3 protein or the peptide. Could the\nauthors add the control obtained by injecting the WT peptide into the buffer?\nThanks for these suggestions. iPTM and pTM scores for AlphaFold3 predictions are\nnow included in the legend of Figure S7D. Additionally, integrated heat peaks for the\ninjection of WT SIM peptide and mutant SIM peptide into buffer are now added as\npanels (D) and (E) respectively of Figure S9. We have clarified the concentrations on\nthe figure and in the legend.\nIn general, as there is no proof that in cells SIM3 SUMOylation is important for meiosis,\nand as the impact of sumoylation of SIM3 on the interaction between Mer2 and Mre11\nis still unclear, the analysis of the impact of sumoylation does not strengthen this study\nand could be moved to the supplementary figures.\nWe respectfully disagree with this comment. The initial version of our manuscript\nalready established that the SIM3 motif of Mre11 works together with the C-terminal\nhelix to promote Mre11 recruitment to chromatin, and that both are important for DSB\nformation.\nNevertheless, to reinforce our claim that SIM3 is relevant for Mre11 recruitment during\nmeiosis, we performed co-immunoprecipitation experiments between Mre2 and Mre11\nin meiotic yeast extracts. These show that mutating the Mre11 SIM3 motif and deleting\nthe C-terminal alpha helix reduce the interaction with Mer2. Importantly, combining the\nmutations abolish the interaction entirely (new data added to Supplementary Figure\n10f). These results are in agreement with our IF, Southern blot, and spore viability data\nthat showed a synergistic effect of these mutations.\nDiscussion:\nIn general, the discussion is based on experimental data that are still to be strengthen.\nWhen discussing about \"the Mer2 alleles previously reported to reduce Mre11\ninteraction that involve amino acids predicted to point inside the Mer2 tetrameric\ncoiled coil\", why should Mer2 stay in its tetrameric state upon binding to Mre11 ?\nThe point we were making is that, based on the AlphaFold model, the C-terminal helix\nof Mre11 engages simultaneously to two adjacent Mer2 helices. Hence, mutating\nresidues inside of the hydrophobic bundle could prevent Mre11 binding by de-\nstabilizing the coiled coil. Hence, we think the mutations we identified more directly\nimpact the interaction than the previously-reported alleles.\nMinor points:\n1. Condensation: in the Mat & Met, the conditions used to study condensation include\nPEG (these conditions are precisely: 25 mM HEPES-HCl (pH 7.5), 2 mM DTT, 1 mg/ml\nBSA, 5% glycerol, 5 mM MgCl2, 5% PEG-3350, and 120 mM NaCl), whereas in the\nlegend of Figure 1E,F, the impact of adding PEG is shown. Is there PEG in the dilution\nbuffer only for the experiments displayed in Figure 1E,F ?\nAll condensation assays included 5% PEG in the reaction buffer, unless mentioned in\nthe legends. Experiments shown in figure 1E, F contained 5% PEG and the control\nreactions were performed without PEG. We have clarified it in the figure legends,\nthank you.\n2. NMR: in the last sentence of the Mat & Met sections, there is an n value that is cited\nbut not present in the equation used for the CSP analysis.\nThank you, the mistake has been corrected.\n3. NMR and ITC: why is the salt concentration so different between the NMR and ITC\nconditions ? The ITC conditions are closer to physiological conditions than the NMR\nconditions. Is the salt concentration influencing the interaction ?\nThank you for pointing that out. Indeed, a lower salt concentration was used for NMR\nto improve signal quality. Higher salt was used for ITC to determine Kd values closer\nto physiological conditions.\nReviewer #3 (Remarks to the Author):\n# NCOMMS-25-61398-T\nRecruitment of Mre11 to recombination sites during meiosis\nIn this manuscript, Priyadarshini et al. reported that a widely studied DSB end\nprocessing factor, Mre11 of the MRX complex, forms condensate at DSB foci in both\nvegetative cells and during meiosis of S cerevisiae, and this involves a C-terminal IDR\nand the last C-terminal 15 amino acids that bind Mer2 and SUMO modification.\nInterestingly, while the condensate is dispensable for vegetative DSB repair, it is\nessential for meiosis. While some results are interesting and may offer new insights\ninto the role of Mre11 in cerevisiae meiosis, the main conclusion lacks strong support\nand significant issues remain unanswered.\nMajor issues:\n1. The biggest issue is the unclearness of the true biological significance of the Mre11\ncondensate in DSB repair.\n• Why the condensate demonstrated different essentialities to vegetative DSB repair\nand meiosis? What is unclear is that if the condensates are part of the DSB foci and\nits formation involved the same protein-protein interaction principles (or same proteins\nand domains are involved), why are they essential for meiosis but not for DSB repair?\nWhat determines the different essentialities here?\nAlthough the domains required for Mre11 foci formation in meiotic and vegetative\nconditions are the same, the assembly of these foci and their composition are\nfundamentally different. Vegetative Mre11 foci depend on DSBs, meiotic ones do not.\nMeiotic Mre11 foci depend on Mer2 and involve a host of other proteins that are absent\nfrom vegetative foci.\nNote that we do not claim that Mre11 condensation is essential in meiosis. We claim\nthat it cannot be essential in vegetative DSB repair (because deleting the IDR\ncompromise condensation in vitro and foci formation in vivo with no detectable effect\non MMS sensitivity).\nOur data is consistent with an essential role for condensation in meiosis, but does not\ndemonstrate that this is the case. Indeed, to show this, one would need a separation-\nof-function mutant that abolishes condensation alone. We do not have such a mutant,\nand finding one will be very difficult, if not impossible.\nWhile we can’t say why Mre11 condensation is dispensable in vegetative conditions,\nbut (perhaps) important during meiosis, we speculate that the self-interactions\nprovided by the Mre11 IDR facilitates its recruitment prior to DSB formation.\n• This also raises additional concerns as to the importance of the Mre11 condensate\nconcept in vegetative DSB repair. If it is not important, then what does it matter, or\nworse, who cares this condensate formation?\nOur results imply that, whatever mechanism drives high local Mre11 accumulation\nfollowing exogenous DNA damage in vegetative cells, it is not essential for DSB repair\nin that context. It is also possible that IDR-dependent focal accumulation might have\nfunctional consequences in DSB repair, but it simply did not manifest in our survival-\nbased assay. Possibly, this accumulation could be a side effect of the self-assembly\nproperty of Mre11 that is important in other context, including meiosis. However, note\nthat Mre11 also has established roles in other cellular contexts beyond exogenous\nDNA damage repair, including checkpoint activation, telomere maintenance,\nstabilizing stalled replication forks, etc. Mre11\ncondensation could potentially influence some of these pathways, though\ninvestigating this is beyond the scope of this study.\nWe have added the above explanation to lines 339-348 of Discussion.\n• Then how to separate this condensate formation from the normal Mre11 foci at DSB\nrepair site in vegetate cells?\nWe are not sure to really understand what the reviewer means here. What are the\nnormal Mre11 foci the reviewer refers to here? The MMS-dependent foci, not MMS-\ninduced? This is unclear.\nPerhaps the reviewer is trying to ask what is the relationship between in vitro\ncondensates and in vivo foci? As in, do the same biochemical principles that allow in\nvitro condensation applies in vivo? Of course, we cannot answer that question. We\nobserve correlations (e.g. dependence on the IDR) but cannot be sure of the extent to\nwhich these assemblies are related.\n• While a DSB repair assay was performed for meiotic cells, it was absent for vegetate\ncells. Only a survival result was shown for vegetate cells expressing Mre11 mutants.\nShould also examine if these condensate deficient Mre11 mutants impact DSB repair\nin vegetate cells.\nWe agree that it would be interesting to analyze the impact of these mutants in\nvegetative DSB repair in more detail. It is possible that these would uncover more\nsubtle roles of the Mre11 IDR. However, this study focuses primarily on the meiotic\nfunction of Mre11, and such physical analysis of DSB repair are beyond the scope of\nthis study.\n• If condensate is essential for meiosis, can you rescue the lethality issue of the strains\nwith Mre11-IDR or C49 deleted? Are there any analyses to see if the Mre11-IDR or\nthe C49 residues alone can form foci in meiotic cells?\nIt is not clear what the reviewer is suggesting. Rescuing the lethality of which strains\nwith Mre11-IDR or C49?\nIn theory, we could have tested whether Mre11-IDR or C49 can form foci in meiotic\ncells. We did not perform this experiment because we do not consider this a very likely\nscenario, because neither IDR alone nor C49 alone form condensates in vitro, even\nat relatively high concentration (i.e. they are necessary but not sufficient for\ncondensation). In addition, although full-length Mre11 binds Mer2 efficiently in a yeast-\ntwo-hybrid assay, the C49 residues alone do not (Supplementary Fig. 6a). This is\nbecause individual C49 peptides have low affinity for Mer2 (Supplementary Fig. 6e).\nPresumably, the affinity of full-length Mre11 is higher because it forms a stable dimer\nthat enhances the interaction.\nWithout clear answers to these questions, the true biological significance of the Mre11\ncondensates at DSB sites remain uncertain.\n2. How exactly Mre11 condensates are formed, regulated, and contributed to DSB foci\nin vegetate and meiotic cells is unclear.\n• First, based on data presented, there are five regions/motifs of Mre11 shown to be\ninvolved: the IDR (~520-677), the C49 (residues 643-692), the C15 (residues 677-\n692), and the SUMO binding region (residues 633-637). The C15 binds to Mer2 and\ndeletion of which caused similar level of foci reduction as the C49 deletion; yet, C15\nis not essential for meiosis essentiality, whereas the C49 does. These results suggest\nthe last 34 residues (643-677) of the IDR are important for meiosis. However, a\ncomprehensive comparison of these above five fragments on in vitro condensate\nassembly with or without Mer2 is lacking. Also, how these fragments contribute to\nMre11 foci formation in vegetate vs meiotic cells are unclear.\nIndeed, recently work (Aithal, 2024) shows that the last 34 residues of Mre11 are\nrequired for interaction with Spo11, as mentioned in the discussion.\nWe agree with the reviewer that each of the mutants and truncations studied in the\nmanuscript need to be systematically characterized. In an effort to complete the\nanalyses of the mutants, we purified eGFP-Mre11-ΔC15 and performed condensation\nassays with and without Mer2. New data is added to Supplementary Fig. 6b-d. Similar\nto eGFP-Mre11-ΔC49, we observed reduced foci intensity and colocalization with\nMer2 foci, indicating that these residues contribute to condensate formation and\ninteraction with Mer2 within condensates.\n• Second, there is the lack of DSB repair in vegetate vs meiotic cells expressing Mre11\nmutants with deletion of just the above five fragments. Similarly, meiotic cell viability\nof these mutants is important to show.\nTo further complete the characterization of each of our mutants, we performed\nimmunofluorescence and MMS sensitivity analyses of Mre11-ΔC15, Mre11-SIM, and\nMre11-ΔC15+SIM strains, which revealed no detectable effect in vegetative\nconditions, as expected. These data are presented in Supplementary Fig. 10g, h.\nYeast spore viability data for each of the mutants are provided in Figure 4C (Mre11-\nWT, Mre11-ΔC49, Mre11-ΔIDR) and Figure 7C (Mre11-WT, Mre11-ΔC15, Mre11-\nSIM, Mre11-ΔC15+SIM), confirming the impact of these fragments in meiosis.\n3. While the manuscript presented some interesting findings, often there was no\ndiscussion regarding the biological/pathological implications/significance of the\nfindings.\n• Fig 3B has no discussion regarding the increase in the percentage of Mre11\naggregates by 1,6-HD treatment and the relationship of this increase to the reduction\nin Mre11 foci.\nLines 327-338 are added to the discussion.\n• Fig 4A showed no Mre11 aggregates by 1,6-HD, which is different from vegetate\ncells and somehow may indicate a difference between these two states. But then, it\ndid so when expressed in meiotic mer2-KRRR cells in Fig 4D. So, going back to the\npoint above, what are exactly the difference between these foci and the large-in-size\naggregates of Mre11? Are these reflecting real biological/pathological changes or just\nexpression-induced nuance of Mre11 under different background? This also ties to the\nmajor issue #1.\nIn vegetative conditions, hexanediol treatment led to hyper-accumulation of Mre11 into\none or a few large aggregates which also mirrored the aggregates observed in a sub-\npopulation of cells harboring mre11-ΔIDR or mer2-KRRR mutations. The reason for\nthis hyperaccumulation remains unclear. It could be a consequence of the self-\nassembly property of Mre11 or the MRX complex, akin to synaptonemal complex\nproteins that form polycomplexes under pathological conditions or may reflect a\nbinding preference of Mre11/MRX to an unknown partner. One way to understand this\nis that, under normal (vegetative or meiotic) conditions, the affinity of Mre11 to its\nsubstrate and cellular partners allow its localization throughout the nucleus. In some\npathological conditions (e.g. upon hexanediol treatment in vegetative cells, or in a\nmer2-KRRR background in meiotic cells), the network of interactions that fine-tunes\nMre11 localization is compromised, leading to its local hyperaccumulation (by self-\nassembly or by tethering to unknown cellular factors).\nNevertheless, they are not due to expression-induced artifacts, as TCA extractions\nshow no obvious changes in Mre11 levels in the mutants strains or upon hexanediol\ntreatment (Figure S4). We have broadly discussed this in lines 327-338 of Discussion.\nMinor issues:\n• Fig 1B & 1D: it stated that “While nuclease treatment strongly reduced the numbers\nof Mre11 and MRX foci, the minority of Mre11 foci that resisted nuclease treatment\ntended to accumulate more protein”. Where is the evidence that the residual\ncondensate accumulated more protein? The selected representative images clearly\ndid not support that. The quantitative intensity data in Fig 1B and 1D showed much\nreduced fluorescence intensities, which reflect the abundance of proteins in any\ncondensate, for all foci (including the nuclease resisted ones). None of the quantitated\nfoci after nuclease treatment retained an intensity level even close to the lowest data\npoint prior to nuclease treatment in these two panels. This statement clearly does not\nhold.\nWe thank the reviewer for pointing this out. To clarify, we quantified intensity per focus\nin both untreated and DNAseI-treated Mre11 condensates, and the data are now\nshown in Supplementary Fig. 1f. The representative image has been updated to better\nreflect these quantifications. We also clarified in the main text that this accumulation\nis observed for Mre11-treated samples, but not MRX.\n• Fig 4F was assembled with equal molar ratio of Mre11 and Mer2, then why a 4-fold\nratio was used for Fig 4G (Mre11-C49:Mer2=4:1)? Will Mre11-C49 still coat Mer2\ncondensates if mixed at 1:1 ratio? In fact, if Mer2 works as a tetramer to recruit the\nMRX complex, it would be Mre11-C49:Mer2=1:4 in these in vitro assembly reactions.\nThis is not correct, since each Mer2 tetramer has four potential Mre11 binding sites\n(Figure S5A). Either way, we are not comparing the results of Figure 4F with Figure\n4G, and a stochiometric assembly is not essential when studying colocalization\nbetween Mre11 and Mer2 condensates because condensates are formed by\nmultivalent, collective interactions that are not dictated by rigid stoichiometry.\n• Fig 5A only modeled the C49 of Mre11. What will happen if a larger domain of Mre11\n(like 620-692 used in Fig S7A) is used? This is important as the authors proposed\nmultiple motifs of Mre11 binding to Mer2. There are a lot of back-and-forth information\nregarding the domains that are involved in Mer2 binding and condensate formation of\nMre11, which were not clearly and systematically evaluated.\nTo clarify, we do not report multiple interaction domains between Mer2 and Mre11.\nWe report (i) a direct protein-protein interaction between the Mre11-C15 helix\n(involving LLK residues) that binds to Mer2-SSM1 (involving EQEK residues), and (ii)\na SUMO-SIM interaction between Mre11-SIM3, and an unidentified SUMOylated\ntarget (that may be Mer2). In addition, we also propose that (iii) Mre11 self-interaction\n(through C-term IDR and C49) may participate in Mre11 recruitment and accumulation\nduring meiosis.\nModelling the Mer2-Mre11 and SUMO-SIM interaction simultaneously is not feasible\nbecause AlphaFold3 doesn’t allow incorporation of SUMO modifications.\n• Fig 7: The importance of the SIM3 motif in Mer2 binding in vivo is lacking. Why not\nperforming similar pulldown assay as in Fig 5B for Mre11-SIM3 mutant and Mer2?\nWithout this information, the model in Fig 7D and the conclusion in the text are\npremature.\nWe disagree with the reviewer. The initial submission presented detailed\ncharacterization of the SIM3 motif in vitro (NMR and ITC, Figure 6) and in vivo (IF,\nSouthern blotting and spore viability, Figure 7). These data indicate that the SIM3 has\na subtle role in meiosis, but collaborates with the C-terminal helix in recruiting Mre11.\nIn the revised manuscript, we further reinforced this conclusion by including co-IP data\n(Supplementary Fig. 10f) that also confirms the synergistic effect of the SIM3 and C15\ndeletion on Mer2 interaction. Therefore, we are confident about our conclusions and\nthe model presented in Figure 7D.\n• Some of the selected representative images are not impressive to support the\nconclusions. For instance, Fig 1A and S1C hardly tell any foci intensity increases.\nFig 2C did not show any reduction in foci number for Mre11Δ290-472. The IDR\ndeletion Mre11 mutant had much weaker foci than the C49 deletion in Fig 4B; yet,\nthe quantitation showed the opposite.\nWe understand that this is somewhat confusing, but please note that the quantification\nin Figure 4B shows foci number, not intensity. The images shown are indeed\nrepresentative of the results, because the foci observed in Mre11-ΔIDR mutants are\nmuch less intense as those observed for Mre11-C49, as described in the text.\n• Figure legends are generally too simplified with much information missing. For\ninstance, what was the conc of the eGFP-Mre11 protein in Fig S1B? What are conc of\nDNA in Fig 1A and 1C? What time point was the image taken for Fig 1A? Do Fig 2C-\nE contain a crowding agent? It would help the readers if all figure legends provided\ndetailed information.\nThe experimental conditions of the condensation assays are detailed in the M&M\nsection. All reactions are performed in identical standard conditions, unless specified.\nWe have verified all the legends and made sure that the relevant information is\nprovided when the experimental condition differed from the standard condition.\n• “…focus formation of Mer2-EQEK was diminished compared to wild-type Mre11 by\napproximately 45% (Figure 5D)”: Fig 5 D has nothing to do with Mer2 foci, but instead,\nit showed Mre11 foci under different backgrounds including in the Mer2-EQEK strain.\nWe thank the reviewer for pointing that out. This mistake has been corrected.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers\nwho co-review manuscripts.\nReferences in support of the point-by-point response:\n1. Hohl, M., Yu, Y., Kuryavyi, V., Patel, D. & Petrini, J. Structure guided functional\nanalysis of theS. cerevisiae Mre11 complex. Nature Communications 16, 7469\n(2025).\n2. Furuse, M. et al. Distinct roles of two separable in vitro activities of yeast Mre11 in\nmitotic and meiotic recombination. The EMBO Journal 17, 6412–6425 (1998).\n3. Käshammer, L. et al. Mechanism of DNA End Sensing and Processing by the\nMre11-Rad50 Complex. Molecular Cell 76, 382-394.e6 (2019).\n4. Nicolas, Y. et al. Molecular insights into the activation of Mre11-Rad50\nendonuclease activity by Sae2/CtIP. Molecular Cell 84, 2223-2237.e4 (2024).\n5. Kissling, V. M. et al. Mre11-Rad50 oligomerization promotes DNA double-strand\nbreak repair. Nat Commun 13, 2374 (2022).\nDear Reviewers,\nWe thank the reviewers for their constructive comments on our manuscript entitled\n“Recruitment of Mre11 to recombination sites during meiosis”. Point-by-point\nresponses are below (reviewer comments are in black, responses are in blue).\nSincerely,\nCorentin Claeys Bouuaert and Priyanka Priyadarshini\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this revision, the authors have satisfactorily answered all my major and minor\npoints.\nThank you.\nReviewer #2 (Remarks to the Author):\nThe authors answered to all my questions, I am fine with their answers, thanks.\nExcept for the addition of the BLI experiments (Suppl. Fig. 6e).\nFor me, it is mandatory to delete this panel. First, I dont see how you can measure\nan affinity of 1 mM by titrating a ligand between 12.5 and 25 uM. Second, several\nessential controls are lacking, as titrating the analyte onto empty biosensors (Nickel-\nbiosensors interact non-specifically with many ligands) and onto His-SUMO coated\nbiosensors. Third, the legend is unclear, but, if, as I understood, the only displayed\ncontrol experiments gave the less bright curves (titration onto the His-SUMO-Mer2\ncoated chip of MBP alone), then there is a huge non specific signal that should be\nsubtracted from the main BLI curve.\nFor all these reasons, I think that this panel has to be removed.\nWe agree with the suggestion of Reviewer 2 and have removed the BLI data in\nSupplementary Fig. 6e.\nReviewer #3 (Remarks to the Author):\nWhile the authors have addressed many technical points, my central concern\nremains largely unaddressed: the manuscript still fails to establish the biological\nsignificance of Mre11 condensates. Below, I focus specifically on this unresolved\nissue and the authors’ responses to it.\nOriginal comment: The biggest issue is the unclearness of the true biological\nsignificance of the Mre11 condensate in DSB repair.\n• Why the condensate demonstrated different essentialities to vegetative DSB repair\nand meiosis? What is unclear is that if the condensates are part of the DSB foci and\nits formation involved the same protein-protein interaction principles (or same\nproteins and domains are involved), why are they essential for meiosis but not for\nDSB repair? What determines the different essentialities here?\nAuthors’ response: Although the domains required for Mre11 foci formation in meiotic\nand vegetative conditions are the same, the assembly of these foci and their\ncomposition are fundamentally different. Vegetative Mre11 foci depend on DSBs,\nmeiotic ones do not. Meiotic Mre11 foci depend on Mer2 and involve a host of other\nproteins that are absent from vegetative foci.\nNote that we do not claim that Mre11 condensation is essential in meiosis. We claim\nthat it cannot be essential in vegetative DSB repair (because deleting the IDR\ncompromise condensation in vitro and foci formation in vivo with no detectable effect\non MMS sensitivity).\nRevision Comment: If the authors do not claim that Mre11 condensation is essential\nin meiosis, then what is the intended biological conclusion regarding these\ncondensates? Under the authors’ own framing, Mre11 condensation is neither\nessential for vegetative DSB repair nor definitively required for meiosis. What, then,\nis the functional significance of these structures?\nAuthors’ response: Our data is consistent with an essential role for condensation in\nmeiosis, but does not demonstrate that this is the case. Indeed, to show this, one\nwould need a separation-of-function mutant that abolishes condensation alone. We\ndo not have such a mutant, and finding one will be very difficult, if not impossible.\nRevision Comment: This response is internally inconsistent with the earlier statement\nthat “we do not claim that Mre11 condensation is essential in meiosis.”\nWhile this reviewer appreciate the difficulty of generating a clean separation-of-\nfunction mutant, the absence of such evidence means that the functional relevance\nof Mre11 condensation remains entirely speculative. This returns directly to the\noriginal critique: what is the biological significance of Mre11 condensates?\nA substantial portion of the manuscript is devoted to describing these condensates.\nYet, without demonstrable functional consequences, these findings remain largely\ndescriptive and fall short of the mechanistic or conceptual advance expected for a\njournal such as Nature Communications.\nAuthors’ response: While we can’t say why Mre11 condensation is dispensable in\nvegetative conditions, but (perhaps) important during meiosis, we speculate that the\nself-interactions provided by the Mre11 IDR facilitates its recruitment prior to DSB\nformation.\nRevision Comment: The authors further speculate that Mre11 condensation may\nfacilitate recruitment prior to DSB formation in meiosis, but this does not address the\nbroader issue.\nIf condensate formation does not affect repair efficiency, survival, or measurable\nrepair outcomes, then it is unclear why this phenomenon matters biologically. At\npresent, the study does not convincingly explain why these condensates are more\nthan an epiphenomenon of Mre11 self-association.\n• This also raises additional concerns as to the importance of the Mre11 condensate\nconcept in vegetative DSB repair. If it is not important, then what does it matter, or\nworse, who cares this condensate formation?\nAuthors’ response: Our results imply that, whatever mechanism drives high local\nMre11 accumulation following exogenous DNA damage in vegetative cells, it is not\nessential for DSB repair in that context. It is also possible that IDR-dependent focal\naccumulation might have functional consequences in DSB repair, but it simply did\nnot manifest in our survival-based assay. Possibly, this accumulation could be a side\neffect of the self-assembly property of Mre11 that is important in other context,\nincluding meiosis. However, note that Mre11 also has established roles in other\ncellular contexts beyond exogenous DNA damage repair, including checkpoint\nactivation, telomere maintenance, stabilizing stalled replication forks, etc. Mre11\ncondensation could potentially influence some of these pathways, though\ninvestigating this is beyond the scope of this study.\nRevision Comment: This response again underscores the central problem: the\nmanuscript does not establish a clear biological function for Mre11 condensates in\nany context tested. Invoking potential roles in other pathways without experimental\nsupport does not resolve this issue.\nAt present, the condensate concept remains insufficiently justified. Without functional\nevidence—either through clear phenotypic consequences, mechanistic linkage, or\ncausal necessity—the biological significance of Mre11 condensation remains\nunclear.\nFollowing the comments of Reviewer 3 on the biological relevance of condensation,\nwe have toned down the Discussion on the same.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers\nwho co-review manuscripts."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In this revision, the authors have satisfactorily answered all my major and minor points.", "reviewer_2": "The authors answered to all my questions, I am fine with their answers, thanks.\nExcept for the addition of the BLI experiments (Suppl. Fig. 6e).\nFor me, it is mandatory to delete this panel. First, I dont see how you can measure an affinity of 1 mM by titrating a ligand between 12.5 and 25 uM. Second, several essential controls are lacking, as titrating the analyte onto empty biosensors (Nickel-biosensors interact non-specifically with many ligands) and onto His-SUMO coated biosensors. Third, the legend is unclear, but, if, as I understood, the only displayed control experiments gave the less bright curves (titration onto the His-SUMO-Mer2 coated chip of MBP alone), then there is a huge non specific signal that should be subtracted from the main BLI curve.\nFor all these reasons, I think that this panel has to be removed.", "reviewer_3": "While the authors have addressed many technical points, my central concern remains largely unaddressed: the manuscript still fails to establish the biological significance of Mre11 condensates. Below, I focus specifically on this unresolved issue and the authors’ responses to it.\nOriginal comment: The biggest issue is the unclearness of the true biological significance of the Mre11 condensate in DSB repair.\n• Why the condensate demonstrated different essentialities to vegetative DSB repair and meiosis? What is unclear is that if the condensates are part of the DSB foci and its formation involved the same protein-protein interaction principles (or same proteins and domains are involved), why are they essential for meiosis but not for DSB repair? What determines the different essentialities here?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear Reviewers,\nWe thank the reviewers for their constructive comments on our manuscript entitled\n“Recruitment of Mre11 to recombination sites during meiosis”. Point-by-point\nresponses are below (reviewer comments are in black, responses are in blue).\nSincerely,\nCorentin Claeys Bouuaert and Priyanka Priyadarshini\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this revision, the authors have satisfactorily answered all my major and minor\npoints.\nThank you.\nReviewer #2 (Remarks to the Author):\nThe authors answered to all my questions, I am fine with their answers, thanks.\nExcept for the addition of the BLI experiments (Suppl. Fig. 6e).\nFor me, it is mandatory to delete this panel. First, I dont see how you can measure\nan affinity of 1 mM by titrating a ligand between 12.5 and 25 uM. Second, several\nessential controls are lacking, as titrating the analyte onto empty biosensors (Nickel-\nbiosensors interact non-specifically with many ligands) and onto His-SUMO coated\nbiosensors. Third, the legend is unclear, but, if, as I understood, the only displayed\ncontrol experiments gave the less bright curves (titration onto the His-SUMO-Mer2\ncoated chip of MBP alone), then there is a huge non specific signal that should be\nsubtracted from the main BLI curve.\nFor all these reasons, I think that this panel has to be removed.\nWe agree with the suggestion of Reviewer 2 and have removed the BLI data in\nSupplementary Fig. 6e.\nReviewer #3 (Remarks to the Author):\nWhile the authors have addressed many technical points, my central concern\nremains largely unaddressed: the manuscript still fails to establish the biological\nsignificance of Mre11 condensates. Below, I focus specifically on this unresolved\nissue and the authors’ responses to it.\nOriginal comment: The biggest issue is the unclearness of the true biological\nsignificance of the Mre11 condensate in DSB repair.\n• Why the condensate demonstrated different essentialities to vegetative DSB repair\nand meiosis? What is unclear is that if the condensates are part of the DSB foci and\nits formation involved the same protein-protein interaction principles (or same\nproteins and domains are involved), why are they essential for meiosis but not for\nDSB repair? What determines the different essentialities here?\nAuthors’ response: Although the domains required for Mre11 foci formation in meiotic\nand vegetative conditions are the same, the assembly of these foci and their\ncomposition are fundamentally different. Vegetative Mre11 foci depend on DSBs,\nmeiotic ones do not. Meiotic Mre11 foci depend on Mer2 and involve a host of other\nproteins that are absent from vegetative foci.\nNote that we do not claim that Mre11 condensation is essential in meiosis. We claim\nthat it cannot be essential in vegetative DSB repair (because deleting the IDR\ncompromise condensation in vitro and foci formation in vivo with no detectable effect\non MMS sensitivity).\nRevision Comment: If the authors do not claim that Mre11 condensation is essential\nin meiosis, then what is the intended biological conclusion regarding these\ncondensates? Under the authors’ own framing, Mre11 condensation is neither\nessential for vegetative DSB repair nor definitively required for meiosis. What, then,\nis the functional significance of these structures?\nAuthors’ response: Our data is consistent with an essential role for condensation in\nmeiosis, but does not demonstrate that this is the case. Indeed, to show this, one\nwould need a separation-of-function mutant that abolishes condensation alone. We\ndo not have such a mutant, and finding one will be very difficult, if not impossible.\nRevision Comment: This response is internally inconsistent with the earlier statement\nthat “we do not claim that Mre11 condensation is essential in meiosis.”\nWhile this reviewer appreciate the difficulty of generating a clean separation-of-\nfunction mutant, the absence of such evidence means that the functional relevance\nof Mre11 condensation remains entirely speculative. This returns directly to the\noriginal critique: what is the biological significance of Mre11 condensates?\nA substantial portion of the manuscript is devoted to describing these condensates.\nYet, without demonstrable functional consequences, these findings remain largely\ndescriptive and fall short of the mechanistic or conceptual advance expected for a\njournal such as Nature Communications.\nAuthors’ response: While we can’t say why Mre11 condensation is dispensable in\nvegetative conditions, but (perhaps) important during meiosis, we speculate that the\nself-interactions provided by the Mre11 IDR facilitates its recruitment prior to DSB\nformation.\nRevision Comment: The authors further speculate that Mre11 condensation may\nfacilitate recruitment prior to DSB formation in meiosis, but this does not address the\nbroader issue.\nIf condensate formation does not affect repair efficiency, survival, or measurable\nrepair outcomes, then it is unclear why this phenomenon matters biologically. At\npresent, the study does not convincingly explain why these condensates are more\nthan an epiphenomenon of Mre11 self-association.\n• This also raises additional concerns as to the importance of the Mre11 condensate\nconcept in vegetative DSB repair. If it is not important, then what does it matter, or\nworse, who cares this condensate formation?\nAuthors’ response: Our results imply that, whatever mechanism drives high local\nMre11 accumulation following exogenous DNA damage in vegetative cells, it is not\nessential for DSB repair in that context. It is also possible that IDR-dependent focal\naccumulation might have functional consequences in DSB repair, but it simply did\nnot manifest in our survival-based assay. Possibly, this accumulation could be a side\neffect of the self-assembly property of Mre11 that is important in other context,\nincluding meiosis. However, note that Mre11 also has established roles in other\ncellular contexts beyond exogenous DNA damage repair, including checkpoint\nactivation, telomere maintenance, stabilizing stalled replication forks, etc. Mre11\ncondensation could potentially influence some of these pathways, though\ninvestigating this is beyond the scope of this study.\nRevision Comment: This response again underscores the central problem: the\nmanuscript does not establish a clear biological function for Mre11 condensates in\nany context tested. Invoking potential roles in other pathways without experimental\nsupport does not resolve this issue.\nAt present, the condensate concept remains insufficiently justified. Without functional\nevidence—either through clear phenotypic consequences, mechanistic linkage, or\ncausal necessity—the biological significance of Mre11 condensation remains\nunclear.\nFollowing the comments of Reviewer 3 on the biological relevance of condensation,\nwe have toned down the Discussion on the same.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers\nwho co-review manuscripts."}]

Decision letter