Recruitment of Mre11 to recombination sites during meiosis

Recruitment of Mre11 to recombination sites during meiosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Recruitment of Mre11 to recombination sites during meiosis Corentin Claeys Bouuaert, Priyanka Priyadarshini, Mahesh Survi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7215871/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The Mre11 nuclease is part of the highly conserved MRX complex involved in the repair of DNA double-strand breaks (DSBs). During meiosis in budding yeast, MRX is also required for the programmed induction of DSBs by Spo11, thereby initiating homologous recombination to promote accurate chromosome segregation. Recruitment of Mre11 to meiotic DSB sites depends on Rec114-Mei4 and Mer2 (RMM), which are thought to organize the meiotic DSB machinery by a mechanism involving biomolecular condensation. Here, we explored the role of Mre11 during meiosis and its relationship to RMM condensation. We show that both Mre11 and MRX complexes form DNA-dependent, hexanediol sensitive condensates in vitro . In vivo , Mre11 assembles into DNA damage-dependent foci in vegetative cells and DSB-independent foci in meiotic cells. In vitro condensates and in vivo foci both depend on the C-terminal intrinsically-disordered region (IDR) of Mre11. Importantly, while the Mre11 IDR is dispensable for vegetative DNA repair it is essential during meiosis. The C-terminal region of Mre11 forms a short α-helix that binds a conserved region of Mer2, and mutating residues within this interface reduces Mre11 foci and DSB formation. Finally, we identified a SUMO-interacting motif within the Mre11 IDR that enhances recruitment of Mre11 during meiosis and facilitates DSB formation. Our results provide new insights into the biophysical properties of Mre11 and its role in initiating meiotic recombination. Biological sciences/Biochemistry/Proteins/DNA-binding proteins Biological sciences/Molecular biology/Cell division/Meiosis Biological sciences/Molecular biology/DNA damage and repair/Double-strand DNA breaks Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 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 death 3 . 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 recombination 4 . 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 shuffling 5 , 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 proteins 7 , 8 . In Saccharomyces cerevisiae , the nine known DSB proteins include Rec114-Mei4 and Mer2. These RMM proteins are proposed to assemble into nucleoprotein condensates along meiotic chromosomes 9 that act as sub-cellular compartments that recruit other DSB proteins to stimulate DNA cleavage by controlling Spo11 dimerization 10 – 12 . This condensation model provides a mechanism that ties DSB formation to the loop-axis organization of meiotic chromosomes 13 , 14 . In S. cerevisiae , DSB formation also depends on the Mre11-Rad50-Xrs2 (MRX) complex 15 – 18 . In addition, MRX initiates the resection of DSB ends in both meiotic and mitotically cycling cells, through the endonuclease and exonuclease activities of Mre11 19–25 . Further, the MRX complex has notable roles in telomere maintenance, stabilization of replication forks, and viral infection 26 – 28 . 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 formation 29 , 30 and was recently found to bind Spo11 31 . 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 microscopy 30,32,33 . In vitro , Mre11-Rad50 complexes have been shown to form higher-order assemblies on DNA, mediated by Rad50 oligomerization 34 . 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 ( Figure S1A and S2A ) 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 ( Figures 1A and 1C ). 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 ( Figure S1B ). Time-course analysis showed a progressive increase in foci number and intensity over time ( Figure S1C ); and protein titration revealed a steep increase in foci intensity above 200 nM Mre11 ( Figure S1D ). A similar concentration-dependent increase in foci intensity was also observed for MRX condensates ( Figure S2B ). Mre11 and MRX condensation was also promoted in the presence of crowding agents ( Figures 1E and 1F ). Mre11 condensation was enhanced in the presence of divalent metal ions, likely through charge neutralization ( Figure S1E ). To address whether the condensates are reversible, we assembled condensates for 20 minutes and treated the reactions with DNase I. 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 ( Figures 1B and 1D ). This observation is consistent with the idea that, following initial nucleation by DNA, Mre11 and MRX 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 condensates 35 . Ten-minute treatment with 5% hexanediol led to a strong reduction in foci intensity ( Figures 1G and 1I ). Similarly, brief exposure to 500 mM NaCl strongly reduced foci intensity ( Figures 1H and 1J ). 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 ( Figures 2A and 2B ) 36–38 . 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 meiotic DSB formation 29 . To identify the domain(s) required for Mre11 condensation, we purified eGFP-tagged truncations ( Figures S3A and S3B ). 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 ( Figure 2C) . In contrast, deleting the disordered region (residues 524-677, Δ IDR ) or C-terminal 49 residues (residues 644-692, Δ C49 ) strongly reduced Mre11 foci intensity ( Figure 2D) . Similarly, the Mre11 C-terminal extremity was also required for efficient focus formation of eGFP-tagged MRX complexes ( Figure 2E ). To understand the effects of Mre11 truncations on DNA-binding activity, we quantified binding to a pUC19 plasmid substrate by gel shift analysis ( Figures S3C and S3D ). All of the truncations retained significant DNA-binding activity. Similar to Mre11 WT , Mre11 Δ10-270 , and Mre11 Δ290-472 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. However, these species of reduced electrophoretic mobility were absent with Mre11 Δ IDR and Mre11 Δ C49 . While the Mre11 IDR is required for efficient Mre11 condensation, purified eGFP-tagged Mre11 IDR alone failed to form condensates and showed reduced DNA binding as compared to the WT or Mre11 Δ IDR ( Figures S3E-S3F ) . 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 contribute 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 exposure to 5% 1,6-hexanediol readily dissolved MMS-induced foci, which instead coalesced into a few aggregates of high intensity ( Figures 3A and 3B) . Deletion of the IDR ( mre11- Δ IDR ) or C-terminal 49 residues ( mre11-ΔC49 ) severely reduced MMS-induced focus formation relative to wild type ( Figure 3C, S4B, S4C ). Despite being important for focus formation, the C-terminus of Mre11 was previously shown to be dispensable for vegetative DNA repair 29 . 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 ( Figure 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 spreads 30,31 . Analogous to mitotic cells, a brief exposure to 1,6-hexanediol disassembles these foci ( Figures 4A and S4A ). 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 allele 39 ( Figures S4D and S4E ). 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 ( Figure S4D ). Consistent with the known meiotic function of the Mre11 C-terminus 29 , mre11-C49 cells produced much fewer foci in meiosis than the wild-type MRE11 cells ( Figure 4B ). However, the few foci observed had an intensity similar to the wild type, suggesting that 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 ( Figure 4B, 4E, S4B, S4C ). As expected, meiosis failed in both mre11-C49 and mre11-ΔIDR strains, producing only dead spores ( Figure 4C ). Meiotic foci of Mre11 depend on most other DSB proteins, including RMM 40 , and direct interactions have been demonstrated between Mre11, Mer2 41 , and Spo11 31 . 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 formation 9 . Similar to mer2Δ strains, the formation of Mre11 foci was strongly reduced in a mer2-KRRR mutant ( Figure 4D ), despite no effect on protein levels ( Figures S4F and S4G ). 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 ( Figures 4D and 4E ). 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 ( Figure 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 ( Figure 4F ), suggesting that the C-terminus of Mre11 is important for interaction with Mer2. While C-terminus of Mre11 does not form condensates independently, in the presence of Mer2, it forms a halo-like structure around Mer2 condensates ( Figure 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 AlphaFold2 42 to model interaction between Mre11-C49 and a Mer2 tetramer (previously defined as the Mer2 oligomeric state 9 ) 43 . AlphaFold produced a model in which the terminal a-helix of Mre11 is positioned close to the N-terminal end of the Mer2 coiled coil ( Figure 5A) . Interestingly, this region contains a conserved sequence motif 1 (SSM1) 44 that was previously implicated in Mre11 binding 41 . 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 ( Figure S5A ). In the AlphaFold model, the Mre11 C-terminal helix binds anti-parallel to the Mer2 coiled coil ( Figure 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 ( Figures S5B and S5D ). 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 ( Figure 5B ). Consistent with the AlphaFold model, alanine mutations of the Mer2-EQEK or Mre11-LLK residues strongly decreased the interaction between the two partners ( Figures 5B and 5C ). To establish the relevance of this interaction for Mre11 focus formation in vivo , we performed immunofluorescence analysis of the Mer2-EQEKmutant protein. Consistent with our in vitro analysis, focus formation of Mer2-EQEK was diminished compared to wild-type Mre11 by approximately 45% ( Figure 5D ). Similarly, deleting the C-terminal a-helix of Mre11 ( mre11-ΔC15 ) also reduced focusformation by approximately 73%. ( Figure 5D ). Importantly, reduced focus formation was not due to reduced levels of Mer2-EQEKor Mre11-ΔC15proteins, which were similar to wild-type Mre11 (Figures S6A-S6C) . 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 ( Figure 5E ), while the mre11-ΔC15 mutation caused reduced and delayed DSB formation ( Figure 5F ), even though the progression of meiosis was unchanged ( Figure S6D ). Consistently, mer2-EQEK mutant spores were completely inviable while the mre11-ΔC15 truncation showed 80% spore viability ( Figures 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 novel SUMO-interaction motif. Several DSB proteins, including Mer2, were shown to be SUMOylated during meiosis, and SUMOylation regulates all aspect of meiotic prophase I, including DSB formation 45 . 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 cells 46 . Using the GPS-SUMO tool 47 , we identified a third potential SIM, here called SIM3 (IIMVS), located towards the end of the Mre11 IDR( Figures 6A and S7A ). 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 SIM 48 ( Figures S7B-S7D ). To validate the Mre11-SIM3, we performed Nuclear Magnetic Resonance (NMR) spectroscopy on purified and isotopically labelled U-[ 13 C, 15 N] yeast Smt3 in the presence of a synthetic Mre11-SIM3 peptide ( Figures 6B and S7E ). As a control, we mutated residues corresponding to Mre11 I633 and M635 to alanine, predicted to contribute to the interaction with Smt3 ( Figure S7B ). 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 ( Figures 6B-6D ). Residues I35-I39 form part of the second b-sheet of Smt3 and R46 is located on the first a-helix that are both predicted to bind the SIM3 peptide ( Figure 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 ( Figure S8 ). To evaluate the affinity of Smt3 for Mre11-SIM3, we performed isothermal titration calorimetry (ITC) binding experiments between the Mre11-SIM3 peptides and Smt3. ITC analysis confirmed that the wild-type SIM3 peptide binds Smt3 with a K D of 20 ± 10 µM, typical for SUMO-SIM interactions 49,50 . In contrast, the mutant SIM3 peptide failed to interact with Smt3 ( Figures 6F and S9 ). 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 ( Figure 7A ), which was associated with delayed and reduced DSB formation ( Figure 7B and S10C ). Combining the SIM3 mutation with truncation of the Mre11 C-terminal a-helix ( mre11-ΔC15+SIM3 ) further reduced Mre11 foci and abolished DSB formation ( Figures 7A and 7B ), while protein level remained unchanged ( Figures S10A and S10B ). Consequently, the mre11-SIM3 mutant had slightly reduced spore viability (86%), while mre11-ΔC15+SIM3 spores were completely inviable ( Figures 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 (Figure S10D) . 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 using the yeast-two-hybrid assay. While truncation of the terminal 15 amino acids of Mre11 abolished the interaction with Mer2, mutation of the SIM3 motif had no discernable impact ( Figure S10E ). We note, however, that Mer2 may not be SUMOylated in this system, which employs mitotically cycling cells. Hence, the potential contribution of Mer2 SUMOylation to productive interaction with Mre11 during meiosis remains unclear. DISCUSSION Budding yeast Mre11 has a C-terminal tail with meiosis-specific functions The MRX complex plays key functions in the maintenance of genomic integrity throughout eukaryotes 51,52 . However, its role in promoting the formation of Spo11-dependent DSBs during meiosis has only been reported in budding yeast and C. elegans 15–17,53 . Indeed, MRX orthologs are not required for meiotic DSB formation in A. thaliana 54 , S. pombe 55 , and mice 56,57 . In C. elegans , MRE-11 and RAD-50 are necessary for DSB formation 53,58 , but the ortholog of Xrs2, NBS-1, is not 59 . In S. cerevisiae , the binding of Mre11 to DSB hotspots depends on the presence of all other DSB proteins except Rad50 40 . This suggests that the MRX complex may be the final component recruited to the DSB machinery and raises the possibility that MRX recruitment may trigger Spo11’s catalytic activity, though the underlying mechanism remains unknown. It has been suggested that the requirement for the MRX complex prior to DSB formation serves to coordinate DSB formation with downstream repair, thereby minimizing genomic instability 40 . Supporting this idea, DSBs detected in wild-type cells are typically fully resected, indicating that processing occurs more rapidly than break accumulation. This observation is consistent with tight coordination between DSB formation and repair 60–63 . It was previously shown that the C-terminus of Mre11 is dispensable for DSB repair in mitotically cycling cells, but essential for the formation of meiotic DSBs 29,40 . Here, we show that this essential meiotic function of the Mre11 C-terminus involves multiple mechanisms that collaborate to promote Mre11 recruitment to recombination sites ( Figure 7D ). The Mre11 C-terminus promotes meiotic DSB formation via multiple mechanisms First, we show that the C-terminal a-helix of Mre11 directly binds the Mer2 SSM1 motif located on the N-terminal side of the tetrameric coiled coil. AlphaFold modeling and mutagenesis identified Mre11-LLK and Mer2-EQEK residues as being responsible for this interaction. Truncating the Mre11 terminal a-helix or mutating the Mer2-EQEK residues in vivo confirmed the functional importance of this interaction for Mre11 foci formation and DSB formation. These data are in line with previous yeast-two-hybrid and pulldown experiments that revealed an interaction between Mre11 and Mer2 41 . We note, however, that the Mer2 alleles previously reported to reduce Mre11 interaction involved amino acids predicted to point inside the Mer2 tetrameric coiled coil, which are therefore likely to affect Mre11 interaction indirectly by compromising the structural stability of Mer2 ( Figure S5C ). Second, we identified and biochemically validated a novel SUMO-interacting motif located within the Mre11 IDR and showed that this motif also participates in Mre11 recruitment and DSB formation during meiosis. While the meiotic phenotypes of the mre11-SIM3 mutant were relatively modest, we find that SIM3 collaborates with the C-terminal a-helix of Mre11 for productive recruitment to precursor DSB sites. Third, a recent study demonstrated that the Mre11 C-terminus is also important for direct interaction with Spo11 31 . While the binding sites were not precisely identified, this interaction implicates Mre11 residues 663 to 676, just upstream of the terminal a-helix. It was noted that a mutant lacking the last 16 amino acids of Mre11 was defective in meiosis despite no effect on Spo11 binding, indicating that interaction with Spo11 is not sufficient for functional recruitment of Mre11. Our data explains this result by demonstrating that the Mre11 terminal a-helix binds to Mer2. Finally, we showed that the C-terminal region of Mre11 is also required to assemble DNA-dependent condensates in vitro . DNA nucleates Mre11 condensates and acts as a scaffold that then recruits free soluble protein through homotypic self-association. We show that the C-terminal 49 residues of Mre11 are sufficient for self-association when Mer2 condensates are provided as a nucleation site, suggesting that this may constitute yet another meiosis-specific function of the Mre11 C-terminal tail. Nevertheless, in the absence of a mutant that specifically abolishes Mre11 self-association, the functional importance of this activity for meiotic DSB formation remains unclear. In summary, the recruitment of Mre11 during meiosis involves at least three, perhaps four, independent functions of the Mre11 C-terminal tail. Assembly of the DSB machinery by hierarchical condensation The in vitro condensation activity of Mre11 and MRX are reminiscent of that of Rec114-Mei4 and Mer2 that also form dynamic and reversible macromolecular assemblies in the presence of DNA 9 . Similar to RMM, Mre11 and MRX condensates likely involve electrostatic interactions between the negatively charged DNA backbone and positively charged residues present in the C-terminal region of Mre11. Condensates are further stabilized by the presence of positively charged ions such as magnesium that presumably inhibit intramolecular repulsion within the DNA substrate. In addition, sensitivity of Mre11 condensates to 1,6-hexanediol indicates that self-association via the low-complexity IDR depends on weak hydrophobic interactions, further supporting the liquid nature of these assemblies. While both Mre11 and MRX condensates share similar properties, they don’t fully mirror each other in their behavior, suggesting that the presence of Rad50 and Xrs2 might confer additional stabilization to MRX condensates, consistent with a previous observation that Rad50 self-interaction drives Mre11-Rad50 oligomerization 34 . The spontaneously assembly of Mer2, Rec114-Mei4 and Mre11/MRX condensates in vitro contrasts with the more stringent assembly of their respective foci in vivo . Indeed, Mer2 foci depend on the axis protein Hop1, Rec114 foci depend on Mer2, and Mre11 foci depend on Mer2 and likely most other DSB proteins 14,40,64–66 . We propose that the DSB machinery assembles via a hierarchical mechanism of successive condensate nucleation and growth events, where Hop1 nucleates the assembly of Mer2 condensates that nucleate Rec114-Mei4 condensates that recruit the Spo11 complex 9 . Mer2 condensates also recruit Mre11 and drive its self-assembly, dependent on stabilizing interactions with Spo11 and other SUMOylated targets ( Figure 7D ). The importance of a SUMO-SIM interaction in Mre11 recruitment If the recruitment of Mre11 constitutes the final step in the assembly of the DSB machinery prior to triggering Spo11-dependent cleavage, SUMOylation of the DSB machinery could serve to mark licensed pre-DSB complexes and/or allow for reversible interactions. What are the SUMOylated targets bound by Mre11 prior to DSB formation? SUMOylation was previously shown to regulate the key events of meiotic prophase I, including DSB formation, and thousands of SUMOylation sites were mapped on meiotic proteins 45 . Amongst those, thirteen sites were identified within Mer2, including several close to the SSM1 motif. Given the physical proximity of the Mre11 LLK and Mer2 EQEK interaction regions and Mre11-SIM3, it is tempting to speculate that SIM3 might be interacting with SUMOylated Mer2 during meiosis, which would presumably serve as an anchor to stabilize the interaction with Mer2. However, the recruitment of Mre11 through the SIM3 motif may involve other SUMOylated DSB proteins, including Spp1, Rec114, Hop1, Red1, and cohesin 45 . MATERIALS AND 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 Table S1 . Plasmids generated in this study were verified by sequencing and are listed in Table S2 . Peptides used were ordered from GenScript or synthesized in the Ballet laboratory and are listed in Table S3 . The expression vector for Mre11 10xHis 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 eGFP Mre11, 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 Xrs2 2xFlag 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 eGFP Mre11 10xHis - Δ10-270 (pry7), eGFP Mre1 10xHis - Δ290-472 (pry5), eGFP Mre1 10xHis -Δ IDR (pry6), and eGFP Mre1 10xHis -Δ C49 (pCCB943) were amplified by inverse PCR from pCCB942 using primers pp26 and pp27, pp28 and pp29, pp24 and pp25, and cb1435 and cb1458, respectively. The amplified product was gel extracted, phosphorylated, and ligated to generate the truncations. The expression vector for eGFP Mre11-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 MBP Mre11-C49 and HisSUMO Mer2 (pCCB1040) was based on a pET-Duet1 vector with the sequence coding for MBP Mre11-C49 cloned within the first position (SacI site) and the sequence coding for HisSUMO Mer2 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 mScarlet Mre11-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. Expression vectors for Alexa594 Mer2 (pCCB750) and eGFP Mer2 (pCCB777) were previously described 9 . The pET28b expression vector for Smt3 (pCCB998) was a kind gift from Chris Lima 67 . 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) and RB268 and RB269 to generate pGAD-C1-Mre11- ΔC15 (pNH1372). 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 × 10 6 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 Mre11 10xHis -Rad50-Xrs2 2xFLAG (MRX) and pCCB942, pCCB866, and pCCB867 were used for the expression of eGFP Mre11 10xHis -Rad50-Xrs2 2xFLAG ( eGFP MRX). Full-length Mre11 10xHis and eGFP Mre11 10xHis were expressed using viruses generated from pCCB856 and pCCB942, respectively. Mre11 truncations eGFP Mre11 10xHis - Δ10-270 , eGFP Mre11 10xHis - Δ290-472 , eGFP Mre11 10xHis - ΔIDR , and eGFP Mre11 10xHis - 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 eGFP Mre11 and eGFP Mre11 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 eGFP MRX complexes and truncations were performed essentially as described 68 . 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 mScarlet Mre11-C49 in E. coli , pry109 was 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 (OD 600 ) of 0.6. Expression was carried out for 20 hours at 16°C with 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) 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. 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). 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 eGFP Mer2 and Alexa594 Mer2 were performed as previously described 9 . For expression of recombinant Smt3 6xHis 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 OD 600 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 20,000 rpm 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-[ 13 C, 15 N] Smt3 6xHis protein, the IPTG induction was carried out in minimal medium containing M9 salts (6.8 g/L Na 2 HPO 4 , 3 g/L KH 2 PO 4 , and 1 g/L NaCl), 2 mM MgSO 4 , 0.2 mM CaCl 2 , trace elements (60 mg/L FeSO 4 ·7H 2 O, 12 mg/L MnCl 2 ·4H 2 O, 8 mg/L CoCl 2 ·6H 2 O, 7 mg/L ZnSO 4 ·7H 2 O, 3 mg/L CuCl 2 ·2H 2 O, 0.2 mg/L H 3 BO 3 , and 50 mg/L EDTA), BME vitamin mix (Sigma), and 1 g/L 15 NH 4 Cl and 2 g/L [ 13 C 6 ]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 MgCl 2 , 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 Alexa488 Mre11 or 100 nM eGFP MRX. 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 mins, 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 script 9 . 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 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 described 43 . 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 (Mer2 iV5 ) using primers dam005 and dam006 on plasmid backbone pMH002 by inverse PCR followed by ligation to generate pDAM003. mer2 iV5 -EQEK was constructed by inverse PCR followed by ligation of pDAM003 using primers cb1451 and cb1562. Genomic integration of mer2-EQEK::HphMX and mer2 iV5 -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 MER2 iV5 ::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 Table S4 . 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 OD 600 0.2 and grown for 12-14 hours at 30°C, 250 rpm. Once the cultures reached OD 600 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 OD 600 0.2 and grown for 3-4 hours to reach OD 600 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 MES-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 H2A-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. 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 (insert company) using 0.5 mm zirconia/silica beads (insert company). 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 described 69 . 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-[ 13 C, 15 N] Smt3 in 20 mM sodium phosphate, 20 mM NaCl (pH 6.5), 0.02% NaN 3 and 10% D 2 O 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 processed in TopSpin 3.6 (Bruker) or NMRPipe 70 and analyzed in CCPNMR 71 . Assignments of Smt3 backbone amide resonances were taken from literature 72 and verified by 3D HNCACB, HN(CO)CACB, and 15 N-edited NOESY-HSQC spectra. Further assignments of methyl resonances were obtained from 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 1 H, 13 C and 15 N 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-[ 13 C, 15 N] Smt3, with the spectral changes monitored in [ 1 H, 15 N] HSQC spectra acquired at each increment. The average chemical shift perturbations (Δδ avg ) were calculated as Δδ avg = (Δδ X 2 /50 + Δδ H 2 /2) 0.5 , where Δδ X and Δδ H are the chemical shift changes of the backbone amide nitrogens or methyl carbons (Δδ X ) and protons (Δδ H ) 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)/H 2 O (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). Declarations Acknowledgments We thank David Alvarez Melo for generating Mer2-iV5 tagged strains, John Weir for plasmids and strains, and CCB laboratory members for discussion. We also thank Biological Imaging facility (IMABIOL) at UCLouvain and Marie-Christine Eloy for providing training in the use of the epifluorescence microscope. This work was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation program (ERC grant agreement 802525 to CCB), and the Fonds National de la Recherche Scientifique (PDR grant T.0031.22 to CCB). PP is funded by FNRS Aspirant fellowships (project 1.A908.22). CCB is a FNRS Research Associate. WEYM and SB acknowledge the Research Council of VUB for support through the Strategic Research Program SRP95 and the infrastructure grant OZR3939. NIH NIGMS grant R01GM074223 supported NH, who is also an Investigator of the Howard Hughes Medical Institute. Author Contributions P.P. designed, executed, and analyzed all experiments except as noted; M. S. performed Southern blot experiments; W.E.Y.M. synthesized peptides under the supervision of S.B., performed ITC experiments and analyzed NMR data; A.N.V. acquired and analyzed NMR data; R.B. performed yeast 2-hybrid experiments under the supervision of N.H.; C.C.B. supervised the research and secured funding. P.P. and C.C.B wrote the paper with input from all authors. Competing interest statement The authors declare no competing interests. References Cooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB j . 2003;17(10):1195-1214. doi:10.1096/fj.02-0752rev De Almeida LC, Calil FA, Machado-Neto JA, Costa-Lotufo LV. DNA damaging agents and DNA repair: From carcinogenesis to cancer therapy. Cancer Genetics . 2021;252-253:6-24. doi:10.1016/j.cancergen.2020.12.002 Tubbs A, Nussenzweig A. Endogenous DNA Damage as a Source of Genomic Instability in Cancer. Cell . 2017;168(4):644-656. doi:10.1016/j.cell.2017.01.002 Oster S, Aqeilan RI. Programmed DNA Damage and Physiological DSBs: Mapping, Biological Significance and Perturbations in Disease States. Cells . 2020;9(8):1870. doi:10.3390/cells9081870 Börner GV, Hochwagen A, MacQueen AJ. Meiosis in budding yeast. GENETICS . 2023;225(2):iyad125. doi:10.1093/genetics/iyad125 Zickler D, Kleckner N. Meiosis: Dances Between Homologs. Annu Rev Genet . 2023;57(1):1-63. doi:10.1146/annurev-genet-061323-044915 Lam I, Keeney S. Mechanism and Regulation of Meiotic Recombination Initiation. Cold Spring Harb Perspect Biol . 2015;7(1):a016634. doi:10.1101/cshperspect.a016634 Yadav VK, Claeys Bouuaert C. Mechanism and Control of Meiotic DNA Double-Strand Break Formation in S. cerevisiae. Front Cell Dev Biol . 2021;9:642737. doi:10.3389/fcell.2021.642737 Claeys Bouuaert C, Pu S, Wang J, et al. DNA-driven condensation assembles the meiotic DNA break machinery. Nature . 2021;592(7852):144-149. doi:10.1038/s41586-021-03374-w Claeys Bouuaert C, Tischfield SE, Pu S, et al. Structural and functional characterization of the Spo11 core complex. Nat Struct Mol Biol . 2021;28(1):92-102. doi:10.1038/s41594-020-00534-w Johnson D, Crawford M, Cooper T, et al. Concerted cutting by Spo11 illuminates meiotic DNA break mechanics. Nature . 2021;594(7864):572-576. doi:10.1038/s41586-021-03389-3 Oger C, Claeys Bouuaert C. SPO11 dimers are sufficient to catalyse DNA double-strand breaks in vitro. Nature . 2025;639(8055):792-799. doi:10.1038/s41586-024-08574-8 Kleckner N. Chiasma formation: chromatin/axis interplay and the role(s) of the synaptonemal complex. Chromosoma . 2006;115(3):175-194. doi:10.1007/s00412-006-0055-7 Panizza S, Mendoza MA, Berlinger M, et al. Spo11-Accessory Proteins Link Double-Strand Break Sites to the Chromosome Axis in Early Meiotic Recombination. Cell . 2011;146(3):372-383. doi:10.1016/j.cell.2011.07.003 Alani E, Padmore R, Kleckner N. Analysis of Wild-Type and rad50 mutants of Yeast Suggests an Intimate Relationship between Meiotic Chromosome Synapsis and Recombination. Cell . 1990;61(3):419-436. doi:10.1016/0092-8674(90)90524-I Ivanov EL, Korolevt VG, Fabre F. Xrs2, a DNA Repair Gene of Saccharomyces Cerevisiae, Is Needed for Meiotic Recombination. GENETICS . 1992;132(3):651-664. doi:10.1093/genetics/132.3.651 Nairz K, Klein F. mre11S —a yeast mutation that blocks double-strand-break processing and permits nonhomologous synapsis in meiosis. Genes Dev . 1997;11(17):2272-2290. doi:10.1101/gad.11.17.2272 Keeney S. Mechanism and control of meiotic recombination initiation. Current Topics in Developmental Biology . 2001;52:1-53. doi:10.1016/S0070-2153(01)52008-6 Moore JK, Haber JE. Cell Cycle and Genetic Requirements of Two Pathways of Nonhomologous End-Joining Repair of Double-Strand Breaks in Saccharomyces cerevisiae . Molecular and Cellular Biology . 1996;16(5):2164-2173. doi:10.1128/MCB.16.5.2164 Paull TT, Gellert M. The 3′ to 5′ Exonuclease Activity of Mre11 Facilitates Repair of DNA Double-Strand Breaks. Molecular Cell . 1998;1(7):969-979. doi:10.1016/S1097-2765(00)80097-0 Garcia V, Phelps SEL, Gray S, Neale MJ. Bidirectional resection of DNA double-strand breaks by Mre11 and Exo1. Nature . 2011;479(7372):241-244. doi:10.1038/nature10515 Casari E, Rinaldi C, Marsella A, et al. Processing of DNA Double-Strand Breaks by the MRX Complex in a Chromatin Context. Front Mol Biosci . 2019;6:43. doi:10.3389/fmolb.2019.00043 Gobbini E, Cassani C, Villa M, Bonetti D, Longhese M. Functions and regulation of the MRX complex at DNA double-strand breaks. Microbial Cell . 2016;3(8):329-337. doi:10.15698/mic2016.08.517 Paull TT. 20 Years of Mre11 Biology: No End in Sight. Molecular Cell . 2018;71(3):419-427. doi:10.1016/j.molcel.2018.06.033 Tisi R, Vertemara J, Zampella G, Longhese MP. Functional and structural insights into the MRX/MRN complex, a key player in recognition and repair of DNA double-strand breaks. Computational and Structural Biotechnology Journal . 2020;18:1137-1152. doi:10.1016/j.csbj.2020.05.013 Lamarche BJ, Orazio NI, Weitzman MD. The MRN complex in double‐strand break repair and telomere maintenance. FEBS Letters . 2010;584(17):3682-3695. doi:10.1016/j.febslet.2010.07.029 Lu R, Zhang H, Jiang YN, Wang ZQ, Sun L, Zhou ZW. Post-Translational Modification of MRE11: Its Implication in DDR and Diseases. Genes . 2021;12(8):1158. doi:10.3390/genes12081158 Oh J, Symington LS. Role of the Mre11 Complex in Preserving Genome Integrity. Genes . 2018;9(12):589. doi:10.3390/genes9120589 Furuse M, Nagase Y, Tsubouchi H, Murakami-Murofushi K, Shibata T, Ohta K. Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination. The EMBO Journal . 1998;17(21):6412-6425. doi:10.1093/emboj/17.21.6412 Usui T, Ohta T, Oshiumi H, Tomizawa J ichi, Ogawa H, Ogawa T. Complex Formation and Functional Versatility of Mre11 of Budding Yeast in Recombination. Cell . 1998;95(5):705-716. doi:10.1016/S0092-8674(00)81640-2 Aithal R, Nangalia K, Spirek M, Chen D, Klein F, Krejci L. Physical interaction with Spo11 mediates the localisation of Mre11 to chromatin in meiosis and promotes its nuclease activity. Nucleic Acids Research . 2024;52(8):4328-4343. doi:10.1093/nar/gkae111 Wen Q, Scorah J, Phear G, Rodgers G, Rodgers S, Meuth M. A Mutant Allele of MRE11 Found in Mismatch Repair-deficient Tumor Cells Suppresses the Cellular Response to DNA Replication Fork Stress in a Dominant Negative Manner. Cohen-Fix O, ed. MBoC . 2008;19(4):1693-1705. doi:10.1091/mbc.e07-09-0975 Tamai T, Reginato G, Ojiri R, et al. Sae2 controls Mre11 endo- and exonuclease activities by different mechanisms. Nat Commun . 2024;15(1):7221. doi:10.1038/s41467-024-51493-5 Kissling VM, Reginato G, Bianco E, et al. Mre11-Rad50 oligomerization promotes DNA double-strand break repair. Nat Commun . 2022;13(1):2374. doi:10.1038/s41467-022-29841-0 Kroschwald S, Maharana S, Simon A. Hexanediol: a chemical probe to investigate the material properties of membrane-less compartments. Matters . Published online May 22, 2017. doi:10.19185/matters.201702000010 Lammens K, Bemeleit DJ, Möckel C, et al. The Mre11:Rad50 Structure Shows an ATP-Dependent Molecular Clamp in DNA Double-Strand Break Repair. Cell . 2011;145(1):54-66. doi:10.1016/j.cell.2011.02.038 Rotheneder M, Stakyte K, Van De Logt E, et al. Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions. Molecular Cell . 2023;83(2):167-185.e9. doi:10.1016/j.molcel.2022.12.003 Petrini J, Hohl M, Yu Y, Kuryavyi V, Patel D. Structure guided functional analysis of the S. cerevisiae Mre11 complex. Published online December 9, 2024. doi:10.21203/rs.3.rs-5390974/v1 Bergerat A, De Massy B, Gadelle D, Varoutas PC, Nicolas A, Forterre P. An atypical topoisomerase II from archaea with implications for meiotic recombination. Nature . 1997;386(6623):414-417. doi:10.1038/386414a0 Borde V, Lin W, Novikov E, Petrini JH, Lichten M, Nicolas A. Association of Mre11p with Double-Strand Break Sites during Yeast Meiosis. Molecular Cell . 2004;13(3):389-401. doi:10.1016/S1097-2765(04)00034-6 Rousová D, Nivsarkar V, Altmannova V, et al. Novel mechanistic insights into the role of Mer2 as the keystone of meiotic DNA break formation. eLife . 2021;10:e72330. doi:10.7554/eLife.72330 Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature . 2021;596(7873):583-589. doi:10.1038/s41586-021-03819-2 Daccache D, De Jonge E, Liloku P, et al. Evolutionary conservation of the structure and function of meiotic Rec114−Mei4 and Mer2 complexes. Genes Dev . 2023;37(11-12):535-553. doi:10.1101/gad.350462.123 Tessé S, Bourbon HM, Debuchy R, et al. Asy2/Mer2: an evolutionarily conserved mediator of meiotic recombination, pairing, and global chromosome compaction. Genes Dev . 2017;31(18):1880-1893. doi:10.1101/gad.304543.117 Bhagwat NR, Owens SN, Ito M, et al. SUMO is a pervasive regulator of meiosis. eLife . 2021;10:e57720. doi:10.7554/eLife.57720 Chen YJ, Chuang YC, Chuang CN, et al. S. cerevisiae Mre11 recruits conjugated SUMO moieties to facilitate the assembly and function of the Mre11-Rad50-Xrs2 complex. Nucleic Acids Res . 2016;44(5):2199-2213. doi:10.1093/nar/gkv1523 Zhao Q, Xie Y, Zheng Y, et al. GPS-SUMO: a tool for the prediction of sumoylation sites and SUMO-interaction motifs. Nucleic Acids Research . 2014;42(W1):W325-W330. doi:10.1093/nar/gku383 Lascorz J, Codina-Fabra J, Reverter D, Torres-Rosell J. SUMO-SIM interactions: From structure to biological functions. Seminars in Cell & Developmental Biology . 2022;132:193-202. doi:10.1016/j.semcdb.2021.11.007 Song J, Zhang Z, Hu W, Chen Y. Small Ubiquitin-like Modifier (SUMO) Recognition of a SUMO Binding Motif. Journal of Biological Chemistry . 2005;280(48):40122-40129. doi:10.1074/jbc.M507059200 Keusekotten K, Bade VN, Meyer-Teschendorf K, et al. Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO. Biochemical Journal . 2014;457(1):207-214. doi:10.1042/BJ20130753 Symington LS. Mechanism and regulation of DNA end resection in eukaryotes. Critical Reviews in Biochemistry and Molecular Biology . 2016;51(3):195-212. doi:10.3109/10409238.2016.1172552 Gnügge R, Symington LS. Keeping it real: MRX–Sae2 clipping of natural substrates. Genes Dev . 2017;31(23-24):2311-2312. doi:10.1101/gad.310771.117 Chin GM, Villeneuve AM. C. elegans mre-11 is required for meiotic recombination and DNA repair but is dispensable for the meiotic G 2 DNA damage checkpoint. Genes Dev . 2001;15(5):522-534. doi:10.1101/gad.864101 Puizina J, Siroky J, Mokros P, Schweizer D, Riha K. Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis. Plant Cell . 2004;16(8):1968-1978. doi:10.1105/tpc.104.022749 Young JA, Hyppa RW, Smith GR. Conserved and nonconserved proteins for meiotic DNA breakage and repair in yeasts. Genetics . 2004;167(2):593-605. doi:10.1534/genetics.103.023762 Liu Y, Lin Z, Yan J, Zhang X, Tong MH. A Rad50 -null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells. Development . 2024;151(8). doi:10.1242/dev.202312 Kim S, Yamada S, Li T, et al. Mouse MRE11-RAD50-NBS1 is needed to start and extend meiotic DNA end resection. Nat Commun . 2025;16(1). doi:10.1038/s41467-025-57928-x Hayashi M, Chin GM, Villeneuve AM. C. elegans germ cells switch between distinct modes of double-strand break repair during meiotic prophase progression. PLOS Genetics . 2007;3(11). doi:https://doi.org/10.1371/journal.pgen.0030191 Girard C, Roelens B, Zawadzki KA, Villeneuve AM. Interdependent and separable functions of Caenorhabditis elegans MRN-C complex members couple formation and repair of meiotic DSBs. Proc Natl Acad Sci USA . 2018;115(19). doi:10.1073/pnas.1719029115 Bishop K, Kleckner N. DMCI: A meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell . 1992;69(3):439-456,. doi:10.1016/0092-8674(92)90446-j Tran PT, Erdeniz N, Dudley S, Liskay RM. Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae. DNA Repair . 2002;1(11):895-912. doi:10.1016/s1568-7864(02)00114-3 Joshi N, Brown MS, Bishop DK, Börner GV. Gradual implementation of the meiotic recombination program via checkpoint pathways controlled by global DSB levels. Molecular Cell . 2015;57(5):797-811. doi:10.1016/j.molcel.2014.12.027 Mimitou EP, Yamada S, Keeney S. A global view of meiotic double-strand break end resection. Science . 2017;335(6320):40-45. doi:10.1126/science.aak9704 Li J, Hooker GW, Roeder GS. Saccharomyces cerevisiae Mer2, Mei4 and Rec114 form a complex required for meiotic double-strand break formation. Genetics . 2006;173(4):1969-1981. doi:10.1534/genetics.106.058768 Henderson KA, Kee K, Maleki S, Santini PA, Keeney S. Cyclin-Dependent Kinase directly regulates initiation of meiotic recombination. Cell . 2006;125(7):1321-1332. doi:10.1016/j.cell.2006.04.039 Maleki S, Neale MJ, Arora C, Henderson KA, Keeney S. Interactions between Mei4, Rec114, and other proteins required for meiotic DNA double-strand break formation in Saccharomyces cerevisiae. Chromosoma . 2007;116(5):471-486. doi:10.1007/s00412-007-0111-y Yunus AA, Lima CD. Purification of SUMO Conjugating Enzymes and Kinetic Analysis of Substrate Conjugation. In: Ulrich HD, ed. SUMO Protocols . Vol 497. Methods in Molecular Biology. Humana Press; 2009:167-186. doi:10.1007/978-1-59745-566-4_11 Cannavo E, Cejka P, Kowalczykowski SC. Relationship of DNA degradation by Saccharomyces cerevisiae Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection. Proc Natl Acad Sci USA . 2013;110(18). doi:10.1073/pnas.1305166110 Murakami H, Borde V, Nicolas A, Keeney S. Gel electrophoresis assays for analyzing DNA double-strand breaks in Saccharomyces cerevisiae at various spatial resolutions. In: Keeney S, ed. Meiosis . Vol 557. Methods in Molecular Biology. Humana Press; 2009:117-142. doi:10.1007/978-1-59745-527-5_9 Delaglio F, Grzesiek S, Vuister GeertenW, Zhu G, Pfeifer J, Bax A. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J Biomol NMR . 1995;6(3). doi:10.1007/BF00197809 Vranken WF, Boucher W, Stevens TJ, et al. The CCPN data model for NMR spectroscopy: Development of a software pipeline. Proteins . 2005;59(4):687-696. doi:10.1002/prot.20449 Sheng W, Liao X. Solution structure of a yeast ubiquitin‐like protein Smt3: The role of structurally less defined sequences in protein–protein recognitions. Protein Science . 2002;11(6):1482-1491. doi:10.1110/ps.0201602 Dosztanyi Z, Csizmok V, Tompa P, Simon I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics . 2005;21(16):3433-3434. doi:10.1093/bioinformatics/bti541 Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryFile.docx Supplementary Information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7215871","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":495276153,"identity":"24b7c619-0480-4857-8f8f-edcbef589cc5","order_by":0,"name":"Corentin Claeys Bouuaert","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-5801-7313","institution":"Université catholique de Louvain","correspondingAuthor":true,"prefix":"","firstName":"Corentin","middleName":"Claeys","lastName":"Bouuaert","suffix":""},{"id":495276154,"identity":"48974896-1aa9-49af-bed1-fff59726632a","order_by":1,"name":"Priyanka Priyadarshini","email":"","orcid":"","institution":"Université catholique de Louvain","correspondingAuthor":false,"prefix":"","firstName":"Priyanka","middleName":"","lastName":"Priyadarshini","suffix":""},{"id":495276155,"identity":"c17d50b4-76d1-4034-a295-75dfba0fa45e","order_by":2,"name":"Mahesh Survi","email":"","orcid":"","institution":"Université catholique de Louvain","correspondingAuthor":false,"prefix":"","firstName":"Mahesh","middleName":"","lastName":"Survi","suffix":""},{"id":495276156,"identity":"12c198ee-6d5c-4aee-aeb9-86305e6448e4","order_by":3,"name":"Wael El Yazidi Mouloud","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Wael","middleName":"El Yazidi","lastName":"Mouloud","suffix":""},{"id":495276157,"identity":"35b64472-5b64-48a8-8cdc-bc1b41c44aa2","order_by":4,"name":"Regina Bohn","email":"","orcid":"","institution":"Howard Hughes Medical Institute / UC Davis","correspondingAuthor":false,"prefix":"","firstName":"Regina","middleName":"","lastName":"Bohn","suffix":""},{"id":495276158,"identity":"312ad5f8-25f5-43a1-98f5-1fac15625d11","order_by":5,"name":"Steven Ballet","email":"","orcid":"https://orcid.org/0000-0003-4123-1641","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"","lastName":"Ballet","suffix":""},{"id":495276159,"identity":"c5f42ccc-a5f3-42b6-b4c5-1ad4faa7f5b9","order_by":6,"name":"Neil Hunter","email":"","orcid":"https://orcid.org/0000-0003-1498-2327","institution":"Howard Hughes Medical Institute / UC Davis","correspondingAuthor":false,"prefix":"","firstName":"Neil","middleName":"","lastName":"Hunter","suffix":""},{"id":495276160,"identity":"f9daee10-4f20-4f7b-9fba-65dce565e32d","order_by":7,"name":"Alexander Volkov","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Volkov","suffix":""}],"badges":[],"createdAt":"2025-07-25 16:01:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7215871/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7215871/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89369089,"identity":"f2e194d8-0cac-48ac-b320-8aa7e867a97e","added_by":"auto","created_at":"2025-08-19 09:50:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":180001,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMre11 and the MRX complex assemble DNA-dependent condensates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, C)\u003c/strong\u003e Effect of the presence of plasmid DNA (pUC19) on condensation of (A) Alexa488-labelled Mre11 and (C) eGFP-tagged MRX. \u003cstrong\u003e(B, D)\u003c/strong\u003e Effect of DNase I on (B) Mre11 and (D) MRX condensates. Condensates were pre-assembled for 20 minutes with 5.7 nM pUC19 prior to challenge. Quantification represents total foci intensity (green) in a field of view and total number of foci per 1000 µm\u003csup\u003e2\u003c/sup\u003e (magenta). Foci intensity is normalized to the mean intensity of samples with DNA for A, C and untreated samples for B, D. Error bars represent mean ± SD from 8-12 fields of view. \u003cstrong\u003e(E, F)\u003c/strong\u003e Effect of the presence of 5% PEG-3350 on (E) Mre11 and (F) MRX condensate formation. Quantification represents total foci intensity (green) in a field of view (normalized to the mean of +PEG images) and total number of foci per 1000 µm\u003csup\u003e2\u003c/sup\u003e (magenta). Error bars represent mean ± SD from 10-12 fields of view. \u003cstrong\u003e(G, I)\u003c/strong\u003e Effect of challenge with 5% 1,6-hexanediol and \u003cstrong\u003e(H, J)\u003c/strong\u003e 0.5 M NaCl on (G, H) Mre11 and (H, J) MRX condensates. Condensates were assembled for 20 minutes prior to challenge and incubated for 15 minutes and 10 minutes, respectively, after challenge. In the control reaction, an equivalent volume (1/20) of water was added instead of hexanediol. Quantification shows foci intensity (normalized to the mean of untreated samples) and total number of foci per 1000 µm\u003csup\u003e2\u003c/sup\u003e (magenta). \u0026nbsp;Error bars represent mean ± SD from 10-12 fields of view.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215871/v1/43326b1343066f3916d09ce0.jpg"},{"id":89369090,"identity":"1bd537d0-7afc-4adb-a7fd-4eda0013f211","added_by":"auto","created_at":"2025-08-19 09:50:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe C-terminal IDR of Mre11 is required for condensation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003e\u0026nbsp;AlphaFold2 predicted model of Mre11 (AF-P32829-F1-v4). \u003cstrong\u003e(B) \u003c/strong\u003eDomain structure of Mre11 and protein disorder prediction using IUPred server\u003csup\u003e73\u003c/sup\u003e. The ANCHOR score predicts the transition probability from a disordered to an ordered structure dependent on a binding partner. \u003cstrong\u003e(C) \u003c/strong\u003eEffect of deleting most of the phosphodiesterase domain (\u003cem\u003eD10-270\u003c/em\u003e), or the capping domain and Rad50-binding domain (\u003cem\u003eD290-472\u003c/em\u003e), \u003cstrong\u003e(D)\u003c/strong\u003e deleting the disordered region (\u003cem\u003eDIDR, \u003c/em\u003eresidues 524-677) and C-terminus (\u003cem\u003eDC49, \u003c/em\u003eresidues 644-692) on Mre11 condensation, and (\u003cstrong\u003eE\u003c/strong\u003e) deleting the C-terminus (\u003cem\u003eDC49\u003c/em\u003e) on MRX condensation. Reactions in (C) and (D) contained 200 nM Mre11. Quantification represents total fluorescence intensity (green) in a field of view and total number of foci per 1000 µm\u003csup\u003e2\u003c/sup\u003e (magenta). Foci intensity is normalized to the mean foci intensity of wild type Mre11 or MRX. Error bars represent mean ± SD from 10-14 fields of view for (C) and (D), and 21 fields of view for (E).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215871/v1/a9a6af6c492af9881f82e6ee.jpg"},{"id":89370214,"identity":"63183e18-94d4-4f8a-bd99-2108db5a3fd7","added_by":"auto","created_at":"2025-08-19 09:58:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Mre11 IDR is required for vegetative foci formation but dispensable for DNA repair.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Effect of 1,6-hexanediol treatment on MMS-induced Mre11\u003csup\u003emyc\u003c/sup\u003e foci visualized by immunofluorescence analysis of vegetatively growing yeast nuclear spreads \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of number of Mre11 foci (left) and Mre11 aggregates (right) before and after 1,6-hexanediol treatment. Error bar shows mean ± SD for at least 25 cells per strain. \u003cstrong\u003e(C)\u003c/strong\u003e Effect of Mre11\u003csup\u003emyc \u003c/sup\u003etruncations on MMS-induced foci formation as visualized by immunofluorescence. Error bars show mean ± SD of Mre11\u003csup\u003emyc\u003c/sup\u003e foci for \u003cem\u003eWT\u003c/em\u003e (n = 51), \u003cem\u003emre11-\u003c/em\u003eΔ\u003cem\u003eIDR \u003c/em\u003e(n = 52), and \u003cem\u003emre11-\u003c/em\u003eΔ\u003cem\u003eC49 \u003c/em\u003e(n = 45). \u003cstrong\u003e(D)\u003c/strong\u003e Sensitivity of wild-type and truncated Mre11\u003csup\u003emyc\u003c/sup\u003e strains to methylmethanesulfonate (MMS) and camptothecin (CPT). Ten-fold serial dilutions from saturated cultures are shown, with dilutions on YPD plates as control.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215871/v1/94caafcaab50d882ce77fdc4.jpg"},{"id":89370215,"identity":"5be7b0a2-7f68-4bad-a51b-ed80411ef05f","added_by":"auto","created_at":"2025-08-19 09:58:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMer2 recruits Mre11 during meiosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Effect of 1,6-hexanediol treatment on meiotic Mre11\u003csup\u003emyc\u003c/sup\u003e foci visualized by immunofluorescence analysis of yeast nuclear spreads 4 hours after transfer to SPM. Quantification shows mean and SD for untreated (n = 53) and 1,6-hexanediol treated cells (n = 73).\u003cstrong\u003e (B, D)\u003c/strong\u003e Immunofluorescence on meiotic nuclear spreads for myc-tagged (B) \u003cem\u003eMRE11-WT\u003c/em\u003e, \u003cem\u003emre11-ΔC49\u003c/em\u003e, \u003cem\u003emre11-ΔIDR\u003c/em\u003e and (D) \u003cem\u003eMRE11-WT\u003c/em\u003e in \u003cem\u003eMER2\u003c/em\u003e, \u003cem\u003emer2Δ\u003c/em\u003e, and \u003cem\u003emer2-KRRR\u003c/em\u003e strains. Quantification shows mean and SD of Mre11\u003csup\u003emyc\u003c/sup\u003e foci for \u003cem\u003eWT\u003c/em\u003e (n = 55), \u003cem\u003emre11-\u003c/em\u003eΔ\u003cem\u003eC49 \u003c/em\u003e(n = 60), \u003cem\u003emre11-\u003c/em\u003eΔ\u003cem\u003eIDR \u003c/em\u003e(n = 53),\u003cem\u003e MER2\u003c/em\u003e (n = 55), \u003cem\u003emer2Δ \u003c/em\u003e(n = 33), and \u003cem\u003emer2-KRRR \u003c/em\u003e(n = 57). \u003cstrong\u003e(C)\u003c/strong\u003e Spore viabilities of strains expressing wild-type or truncated Mre11\u003csup\u003emyc\u003c/sup\u003e. At least 22 tetrads were dissected for each strain (n ³ 88 spores). (\u003cstrong\u003eE\u003c/strong\u003e) Quantification of sub-population of cells showing discrete Mre11 foci and aggregated Mre11 foci in indicated strains. Images used for analysis were from the same experiments in (B) and (D). \u003cstrong\u003e(F)\u003c/strong\u003e Colocalization of fluorescently-labelled Mer2 with wild-type or truncated Mre11. Reactions containing 200 nM of \u003csup\u003eAlexa594\u003c/sup\u003eMer2 and \u003csup\u003eeGFP\u003c/sup\u003eMre11 were assembled separately for 10 minutes then mixed at 1:1 ratio for 30 minutes prior to imaging. Quantification shows the fraction of Mre11 foci overlapping Mer2 foci (white) in a field of view, normalized to Mre11-WT. Error bars represent mean ± SD from 10-15 fields of view. \u003cstrong\u003e(G) \u003c/strong\u003eColocalization of fluorescently-labelled Mre11-C49 and Mer2. Condensates were assembled by mixing 800 nM of \u003csup\u003emScarlet\u003c/sup\u003eMre11-C49 and 200 nM of \u003csup\u003eeGFP\u003c/sup\u003eMer2 for 30 minutes prior to imaging. Controls reactions contained the same concentration but lacked either \u003csup\u003eeGFP\u003c/sup\u003eMer2 or \u003csup\u003emScarlet\u003c/sup\u003eMre11-C49, respectively.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215871/v1/2f07d03881ebcaf076aeee51.jpg"},{"id":89369093,"identity":"af5f29b0-de15-4f64-8d74-18af4aa2692f","added_by":"auto","created_at":"2025-08-19 09:50:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":153736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe C-terminus of Mre11 binds a conserved motif of Mer2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e AlphaFold2 predicted model of Mre11 C-terminal residues (644-692, Mre11-C49) (orange) and Mer2 (41-100) (blue), aligned to the Mer2 coiled-coil domain (residues 41-247) (grey). Magnified-view shows amino acid residues at the predicted interaction surface, Mre11 residues L683, L686, and K690 (LLK) and Mer2 residues E50, Q54, E57, and K61 (EQEK). \u003cstrong\u003e(B)\u003c/strong\u003e Co-expression based pulldown assay between wild type and mutant \u003csup\u003eMBP\u003c/sup\u003eMre11-C49 (prey) and \u003csup\u003eHisSUMO\u003c/sup\u003eMer2 (bait). The LLK and EQEK mutants have the respective residues mutated to alanine. Asterisk (*) denotes free MBP. \u003cstrong\u003e(C)\u003c/strong\u003e Quantification of anti-MBP elution/input signal from pulldown in panel B, normalized to the wild type. Error bars represent mean ± SD from at least seven independent samples. \u003cstrong\u003e(D)\u003c/strong\u003e Immunofluorescence on meiotic nuclear spreads of Mre11\u003csup\u003emyc\u003c/sup\u003e in WT, \u003cem\u003emer2-EQEK\u003c/em\u003e, and \u003cem\u003emre11-\u003c/em\u003eΔ\u003cem\u003eC15 \u003c/em\u003estrains. Quantification of Mre11\u003csup\u003emyc \u003c/sup\u003efoci in WT (n = 54), \u003cem\u003emer2-EQEK \u003c/em\u003e(n = 36), and \u003cem\u003emre11-\u003c/em\u003eΔ\u003cem\u003eC15 \u003c/em\u003e(n = 38). \u003cstrong\u003e(E, F)\u003c/strong\u003e Southern blot analysis of meiotic DSB formation at the \u003cem\u003eGAT1\u003c/em\u003e hotspot. Quantification of panel F show mean and range from n = 2 experiments. \u003cstrong\u003e(G)\u003c/strong\u003e Spore viabilities of wild-type and mutant strains (n ³ 88 spores).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215871/v1/965a1afebcec98ffb3a4aaca.jpg"},{"id":89369092,"identity":"05611334-7ac8-417a-8771-ad3fd81a66e7","added_by":"auto","created_at":"2025-08-19 09:50:08","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":99179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Mre11 C-terminus contains a novel SUMO-interaction motif.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic representation of SUMO-interacting motif (SIMs) in Mre11. \u003cstrong\u003e(B, C)\u003c/strong\u003e Average backbone amide chemical shift perturbations (Δδ\u003csub\u003eavg\u003c/sub\u003e) of Smt3 in the presence of 1.2 molar equivalents of (B) wild type or (C) mutant SIM peptide (sequences indicated). The secondary structure of Smt3 is shown above the plots. The horizontal lines in (B) correspond to the average Δδ\u003csub\u003eavg\u003c/sub\u003e (avg) plus one or two standard deviations (stdev). \u003cstrong\u003e(D)\u003c/strong\u003e Overlaid [\u003csup\u003e1\u003c/sup\u003eH,\u003csup\u003e15\u003c/sup\u003eN] HSQC spectra of Smt3 in its free form (black) or with 1.2 molar equivalents of wild type (green) or mutant (pink) SIM3 peptides. Blue arrows indicate residues that experience highest chemical shift. \u003cstrong\u003e(E)\u003c/strong\u003e Chemical shift mapping of the wild-type SIM peptide binding. Smt3 is shown as (top) cartoon and (bottom) molecular surface coloured by the Δδ\u003csub\u003eavg\u003c/sub\u003e values (pink: Δδ\u003csub\u003eavg\u003c/sub\u003e \u0026gt; avg + stdev; violet: Δδ\u003csub\u003eavg\u003c/sub\u003e \u0026gt; avg + 2*stdev). The bound SIM peptide is in green. The disordered regions at Smt3 N- and C-termini are omitted for clarity. \u003cstrong\u003e(F)\u003c/strong\u003e Normalized heat per peak ΔH (kcal/mol) as a function of molar ratio (peptide/protein concentration) measured by isothermal titration calorimetry upon titration of wild-type (green) or mutant (pink) SIM peptide on Smt3. The solid line shows the best fit to a single binding site model for the wild-type peptide.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215871/v1/8fcb5dff8616577460869129.jpg"},{"id":89370544,"identity":"83d3abf1-e0e8-485e-b3e9-ee33b4ceb7b1","added_by":"auto","created_at":"2025-08-19 10:06:08","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":125579,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA SUMO-SIM interaction fosters the recruitment of Mre11 during meiosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Immunofluorescence on meiotic nuclear spreads of myc-tagged \u003cem\u003eMRE11-WT\u003c/em\u003e, \u003cem\u003emre11-SIM3\u003c/em\u003e, and \u003cem\u003emre11-SIM3+ΔC15 \u003c/em\u003ecells. Quantification of Mre11\u003csup\u003emyc \u003c/sup\u003efoci show mean ± SD of n = 50 cells per strain. \u003cstrong\u003e(B)\u003c/strong\u003e Southern blot analysis of meiotic DSB formation at \u003cem\u003eGAT1\u003c/em\u003e hotspot. Quantifications show mean ± range of n = 2 independent experiments. \u003cstrong\u003e(C) \u003c/strong\u003eSpore viabilities of wild-type and mutant \u003cem\u003eMRE11\u003c/em\u003e strains (n ³ 88 spores). \u003cstrong\u003e(D)\u003c/strong\u003e Schematic model of the mechanisms that drive Mre11 recruitment during meiosis. Mre11 undergoes DNA-dependent condensation, driven by its IDR, and can mingle with Mer2 to form joint condensates. Mre11 is recruited to the DSB machinery via its C-terminus through a SUMO-SIM interaction and direct binding with Spo11 and Mer2.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215871/v1/cdb3a36b49e6b043eda0d619.jpg"},{"id":89371613,"identity":"5d494099-633f-4fb9-84a6-f3dee6879fb1","added_by":"auto","created_at":"2025-08-19 10:14:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2778135,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7215871/v1/665e7278-c216-4a7a-be9c-69a806681dfe.pdf"},{"id":89369096,"identity":"ed330ce8-14a2-4462-b58a-320c4b9bc80e","added_by":"auto","created_at":"2025-08-19 09:50:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7374513,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7215871/v1/2e5ad9666df0a40efde04fbe.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Recruitment of Mre11 to recombination sites during meiosis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCells continuously experience exogenous and endogenous stress leading to cytotoxic lesions such as DNA double-strand breaks (DSBs)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. If not repaired promptly and accurately, DSBs can compromise genome stability resulting in cellular dysfunction, tumorigenesis, or cell death\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. 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 recombination\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDuring 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 shuffling\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. 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 proteins\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, the nine known DSB proteins include Rec114-Mei4 and Mer2. These RMM proteins are proposed to assemble into nucleoprotein condensates along meiotic chromosomes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e that act as sub-cellular compartments that recruit other DSB proteins to stimulate DNA cleavage by controlling Spo11 dimerization\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This condensation model provides a mechanism that ties DSB formation to the loop-axis organization of meiotic chromosomes\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eS. cerevisiae\u003c/em\u003e, DSB formation also depends on the Mre11-Rad50-Xrs2 (MRX) complex\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In addition, MRX initiates the resection of DSB ends in both meiotic and mitotically cycling cells, through the endonuclease and exonuclease activities of Mre11\u003csup\u003e19\u0026ndash;25\u003c/sup\u003e. Further, the MRX complex has notable roles in telomere maintenance, stabilization of replication forks, and viral infection\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhile 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 \u003cem\u003ein vitro\u003c/em\u003e dependent on its C-terminal disordered tail. The Mre11 C-terminus was previously shown to be required for meiotic DSB formation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and was recently found to bind Spo11\u003csup\u003e31\u003c/sup\u003e. 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.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eMre11 and the MRX complex assemble DNA-dependent condensates \u003cem\u003ein vitro\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring meiosis and in mitotically cycling cells exposed to DNA damage, budding yeast Mre11 forms numerous foci visible by immunofluorescence microscopy\u003csup\u003e30,32,33\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e, Mre11-Rad50 complexes have been shown to form higher-order assemblies on DNA, mediated by Rad50 oligomerization\u003csup\u003e34\u003c/sup\u003e. However, whether Mre11 itself participates in the assembly of higher-order structures has not been established.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo 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 (\u003cstrong\u003eFigure S1A and S2A\u003c/strong\u003e) and asked whether they undergo condensation \u003cem\u003ein vitro\u003c/em\u003e. In the presence of plasmid DNA, we found that Mre11 and MRX form large clusters visible by fluorescence microscopy (\u003cstrong\u003eFigures 1A and 1C\u003c/strong\u003e). 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 (\u003cstrong\u003eFigure S1B\u003c/strong\u003e). Time-course analysis showed a progressive increase in foci number and intensity over time (\u003cstrong\u003eFigure S1C\u003c/strong\u003e); and protein titration revealed a steep increase in foci intensity above 200 nM Mre11 (\u003cstrong\u003eFigure S1D\u003c/strong\u003e). A similar concentration-dependent increase in foci intensity was also observed for MRX condensates (\u003cstrong\u003eFigure S2B\u003c/strong\u003e). Mre11 and MRX condensation was also promoted in the presence of crowding agents (\u003cstrong\u003eFigures 1E and 1F\u003c/strong\u003e). Mre11 condensation was enhanced in the presence of divalent metal ions, likely through charge neutralization (\u003cstrong\u003eFigure S1E\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo address whether the condensates are reversible, we assembled condensates for 20 minutes and treated the reactions with DNase I. 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 (\u003cstrong\u003eFigures 1B and 1D\u003c/strong\u003e). This observation is consistent with the idea that, following initial nucleation by DNA, Mre11 and MRX condensates grow primarily through protein-protein interactions.\u003c/p\u003e\n\u003cp\u003eTo further test this interpretation, we asked whether Mre11 and MRX condensates are sensitive to 1,6-hexanediol, an aliphatic alcohol that often dissolves biomolecular condensates\u003csup\u003e35\u003c/sup\u003e. Ten-minute treatment with 5% hexanediol led to a strong reduction in foci intensity (\u003cstrong\u003eFigures 1G and 1I\u003c/strong\u003e). Similarly, brief exposure to 500 mM NaCl strongly reduced foci intensity (\u003cstrong\u003eFigures 1H and 1J\u003c/strong\u003e). Hence, Mre11 and MRX nucleoprotein condensates are stabilized through a combination of ionic and weak hydrophobic interactions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe C-terminal IDR of Mre11 is required for condensation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMre11 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 (\u003cstrong\u003eFigures 2A and 2B\u003c/strong\u003e)\u003csup\u003e36–38\u003c/sup\u003e. 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 meiotic DSB formation\u003csup\u003e29\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo identify the domain(s) required for Mre11 condensation, we purified eGFP-tagged truncations (\u003cstrong\u003eFigures S3A and S3B\u003c/strong\u003e). 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 (\u003cstrong\u003eFigure 2C)\u003c/strong\u003e. In contrast, deleting the disordered region (residues 524-677, Δ\u003cem\u003eIDR\u003c/em\u003e) or C-terminal 49 residues (residues 644-692, Δ\u003cem\u003eC49\u003c/em\u003e) strongly reduced Mre11 foci intensity (\u003cstrong\u003eFigure 2D)\u003c/strong\u003e. Similarly, the Mre11 C-terminal extremity was also required for efficient focus formation of eGFP-tagged MRX complexes (\u003cstrong\u003eFigure 2E\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo understand the effects of Mre11 truncations on DNA-binding activity, we quantified binding to a pUC19 plasmid substrate by gel shift analysis (\u003cstrong\u003eFigures S3C and S3D\u003c/strong\u003e). All of the truncations retained significant DNA-binding activity. Similar to Mre11\u003csup\u003eWT\u003c/sup\u003e, Mre11\u003cem\u003e\u003csup\u003eΔ10-270\u003c/sup\u003e,\u003c/em\u003e and Mre11\u003cem\u003e\u003csup\u003eΔ290-472\u003c/sup\u003e\u003c/em\u003e produced fast-migrating complexes at concentrations up to\u0026nbsp;~25 nM protein, then formed complexes of reduced mobility at higher protein concentration, presumably indicative of higher-order assemblies. However, these species of reduced electrophoretic mobility were absent with Mre11\u003csup\u003eΔ\u003cem\u003eIDR\u003c/em\u003e\u003c/sup\u003e and Mre11\u003csup\u003eΔ\u003cem\u003eC49\u003c/em\u003e\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile the Mre11 IDR is required for efficient Mre11 condensation, purified eGFP-tagged Mre11\u003csup\u003eIDR\u003c/sup\u003e alone failed to form condensates and showed reduced DNA binding as compared to the WT or Mre11\u003csup\u003eΔ\u003cem\u003eIDR\u003c/em\u003e\u003c/sup\u003e(\u003cstrong\u003eFigures S3E-S3F\u003c/strong\u003e)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eWe conclude that, while protein-DNA interactions are important for the formation of Mre11 foci \u003cem\u003ein vitro\u003c/em\u003e, multivalent protein-protein interaction through low-complexity regions contribute to condensation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn mitotically cycling cells, the Mre11 IDR is required for focus formation but dispensable for DNA repair.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 exposure to 5% 1,6-hexanediol readily dissolved MMS-induced foci, which instead coalesced into a few aggregates of high intensity (\u003cstrong\u003eFigures 3A and 3B)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDeletion of\u0026nbsp;the IDR (\u003cem\u003emre11-\u003c/em\u003eΔ\u003cem\u003eIDR\u003c/em\u003e) or C-terminal 49 residues (\u003cem\u003emre11-ΔC49\u003c/em\u003e) severely reduced MMS-induced focus formation relative to wild type (\u003cstrong\u003eFigure 3C, S4B, S4C\u003c/strong\u003e). Despite being important for focus formation, the C-terminus of Mre11 was previously shown to be dispensable for vegetative DNA repair\u003csup\u003e29\u003c/sup\u003e. Consistently, the \u003cem\u003emre11-ΔC49\u003c/em\u003e mutant was resistant to treatment with MMS or camptothecin (CPT). Similarly, \u003cem\u003emre11-ΔIDR\u003c/em\u003e mutants were as resistant to MMS or CPT as wild-type cells (\u003cstrong\u003eFigure 3D\u003c/strong\u003e). Hence, the formation of condensate-like Mre11 foci is dispensable for DSB repair in mitotically cycling cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMer2 condensates directly recruit Mre11 during meiosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring meiosis, Mre11 appears as discrete foci in immunofluorescence staining of prophase I chromosome spreads\u003csup\u003e30,31\u003c/sup\u003e. Analogous to mitotic cells, a brief exposure to 1,6-hexanediol disassembles these foci (\u003cstrong\u003eFigures 4A and S4A\u003c/strong\u003e). However, unlike vegetative conditions, we did not detect large Mre11 aggregates upon 1,6-hexanediol treatment during meiosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile 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 \u003cem\u003espo11-Y135F\u0026nbsp;\u003c/em\u003eallele\u003csup\u003e39\u003c/sup\u003e (\u003cstrong\u003eFigures S4D and S4E\u003c/strong\u003e). In a wild-type background, the number of Mre11 foci tended to decrease in late meiotic prophase, while foci accumulated in a \u003cem\u003espo11-Y135F\u0026nbsp;\u003c/em\u003emutant (\u003cstrong\u003eFigure S4D\u003c/strong\u003e). Consistent with the known meiotic function of the Mre11 C-terminus\u003csup\u003e29\u003c/sup\u003e, \u003cem\u003emre11-C49\u003c/em\u003e cells produced much fewer foci in meiosis than the wild-type \u003cem\u003eMRE11\u0026nbsp;\u003c/em\u003ecells (\u003cstrong\u003eFigure 4B\u003c/strong\u003e). However, the few foci observed had an intensity similar to the wild type, suggesting that the Mre11 C-terminus is important for the nucleation of Mre11 foci, but not their growth. In contrast, the \u003cem\u003emre11-ΔIDR\u003c/em\u003e strain produced fewer and much weaker foci. Approximately 40% of the cells also showed accumulation of fluorescent signal in discrete aggregates (\u003cstrong\u003eFigure 4B, 4E, S4B, S4C\u003c/strong\u003e). As expected, meiosis failed in both \u003cem\u003emre11-C49\u003c/em\u003e and \u003cem\u003emre11-ΔIDR\u003c/em\u003e strains, producing only dead spores (\u003cstrong\u003eFigure 4C\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMeiotic foci of Mre11 depend on most other DSB proteins, including RMM\u003csup\u003e40\u003c/sup\u003e, and direct interactions have been demonstrated between Mre11, Mer2\u003csup\u003e41\u003c/sup\u003e, and Spo11\u003csup\u003e31\u003c/sup\u003e. 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 (\u003cem\u003emer2-KRRR\u003c/em\u003e) that compromises Mer2 focus formation \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e and abolishes DSB formation\u003csup\u003e9\u003c/sup\u003e. Similar to \u003cem\u003emer2Δ\u003c/em\u003e strains, the formation of Mre11 foci was strongly reduced in a \u003cem\u003emer2-KRRR\u003c/em\u003e mutant (\u003cstrong\u003eFigure 4D\u003c/strong\u003e), despite no effect on protein levels (\u003cstrong\u003eFigures S4F and S4G\u003c/strong\u003e). In \u003cem\u003emer2Δ\u003c/em\u003e and \u003cem\u003emer2-KRRR\u003c/em\u003e backgrounds, Mre11 localization was analogous to the \u003cem\u003emre11-ΔIDR\u003c/em\u003e mutant, with few foci of low intensity and accumulation within a large aggregate (\u003cstrong\u003eFigures 4D and 4E\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo test whether Mer2 condensates can directly recruit Mre11 \u003cem\u003ein vitro\u003c/em\u003e, 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 (\u003cstrong\u003eFigure 4F\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen these experiments were performed with the Mre11-ΔC49 truncation and wild-type Mer2, we observed strongly reduced colocalization as compared to wild-type Mre11 (\u003cstrong\u003eFigure 4F\u003c/strong\u003e), suggesting that the C-terminus of Mre11 is important for interaction with Mer2. While C-terminus of Mre11 does not form condensates independently, in the presence of Mer2, it forms a halo-like structure around Mer2 condensates (\u003cstrong\u003eFigure 4G\u003c/strong\u003e). 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe C-terminus of Mre11 binds a conserved motif of Mer2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delve deeper into the interaction between Mre11 and Mer2, we used AlphaFold2\u003csup\u003e42\u003c/sup\u003e to model interaction between Mre11-C49 and a Mer2 tetramer (previously defined as the Mer2 oligomeric state\u003csup\u003e9\u003c/sup\u003e)\u003csup\u003e43\u003c/sup\u003e. AlphaFold produced a model in which the terminal\u0026nbsp;a-helix of Mre11 is positioned close to the N-terminal end of the Mer2 coiled coil (\u003cstrong\u003eFigure 5A)\u003c/strong\u003e. Interestingly, this region contains a conserved sequence motif 1 (SSM1)\u003csup\u003e44\u003c/sup\u003e that was previously implicated in Mre11 binding\u003csup\u003e41\u003c/sup\u003e.\u0026nbsp;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 (\u003cstrong\u003eFigure S5A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn the AlphaFold model, the Mre11 C-terminal helix binds anti-parallel to the Mer2 coiled coil (\u003cstrong\u003eFigure 5A\u003c/strong\u003e). 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\u0026nbsp;Saccharomycetaceae (\u003cstrong\u003eFigures S5B and S5D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo test this model, we co-expressed MBP-tagged Mre11-C49 peptide and HisSUMO-tagged Mer2 in \u003cem\u003eE. coli\u003c/em\u003e and quantified protein interactions by NiNTA pulldown. While the interaction was relatively weak, anti-MBP immunoblot analysis confirmed that Mre11-C49 directly binds Mer2 (\u003cstrong\u003eFigure 5B\u003c/strong\u003e). Consistent with the AlphaFold model, alanine mutations of the Mer2-EQEK or Mre11-LLK residues strongly decreased the interaction between the two partners (\u003cstrong\u003eFigures 5B and 5C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo establish the relevance of this interaction for Mre11 focus formation \u003cem\u003ein vivo\u003c/em\u003e, we performed immunofluorescence analysis of the Mer2-EQEKmutant protein. Consistent with our \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eanalysis, focus formation of Mer2-EQEK was diminished compared to wild-type Mre11 by approximately 45% (\u003cstrong\u003eFigure 5D\u003c/strong\u003e). Similarly, deleting the C-terminal\u0026nbsp;a-helix of Mre11 (\u003cem\u003emre11-ΔC15\u003c/em\u003e) also reduced focusformation by approximately 73%. (\u003cstrong\u003eFigure 5D\u003c/strong\u003e). Importantly, reduced focus formation was not due to reduced levels of Mer2-EQEKor Mre11-ΔC15proteins, which were similar to wild-type Mre11 \u003cstrong\u003e(Figures S6A-S6C)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo address whether the interaction between Mer2 and Mre11 is important for their meiotic function, we analyzed DSB formation at the \u003cem\u003eGAT1\u003c/em\u003e hotspot by Southern blotting. Similar to the \u003cem\u003emre11-ΔC49\u003c/em\u003e mutant, little or no DSB signal was detected in \u003cem\u003emer2-EQEK\u0026nbsp;\u003c/em\u003ecells (\u003cstrong\u003eFigure 5E\u003c/strong\u003e), while the \u003cem\u003emre11-ΔC15\u0026nbsp;\u003c/em\u003emutation caused reduced and delayed DSB formation (\u003cstrong\u003eFigure 5F\u003c/strong\u003e), even though the progression of meiosis was unchanged (\u003cstrong\u003eFigure S6D\u003c/strong\u003e). Consistently, \u003cem\u003emer2-EQEK\u003c/em\u003e mutant spores were completely inviable while the \u003cem\u003emre11-ΔC15\u003c/em\u003e truncation showed 80% spore viability (\u003cstrong\u003eFigures 5G\u003c/strong\u003e). Since \u003cem\u003emre11-ΔC15\u0026nbsp;\u003c/em\u003edoes not phenocopy the \u003cem\u003emre11-ΔC49\u0026nbsp;\u003c/em\u003eand \u003cem\u003emer2-EQEK\u003c/em\u003e 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Mre11 C-terminus contains a novel SUMO-interaction motif.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral DSB proteins, including Mer2, were shown to be SUMOylated during meiosis, and SUMOylation regulates all aspect of meiotic prophase I, including DSB formation\u003csup\u003e45\u003c/sup\u003e. Notably, SUMOylation sites were mapped in the SSM1 region of Mer2 that also harbors the EQEK residues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMre11 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 cells\u003csup\u003e46\u003c/sup\u003e. Using the GPS-SUMO tool\u003csup\u003e47\u003c/sup\u003e, we identified a third potential SIM, here called SIM3 (IIMVS), located towards the end of the Mre11 IDR(\u003cstrong\u003eFigures 6A and S7A\u003c/strong\u003e). 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 \u003cem\u003ebona fide\u003c/em\u003e SIM\u003csup\u003e48\u003c/sup\u003e (\u003cstrong\u003eFigures S7B-S7D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo validate the Mre11-SIM3, we performed Nuclear Magnetic Resonance (NMR) spectroscopy on purified and isotopically labelled U-[\u003csup\u003e13\u003c/sup\u003eC, \u003csup\u003e15\u003c/sup\u003eN] yeast Smt3 in the presence of a synthetic Mre11-SIM3 peptide (\u003cstrong\u003eFigures 6B and S7E\u003c/strong\u003e). As a control, we mutated residues corresponding to Mre11 I633 and M635 to alanine, predicted to contribute to the interaction with Smt3 (\u003cstrong\u003eFigure S7B\u003c/strong\u003e). 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 (\u003cstrong\u003eFigures 6B-6D\u003c/strong\u003e). Residues I35-I39 form part of the second\u0026nbsp;b-sheet of Smt3 and R46 is located on the first\u0026nbsp;a-helix that are both predicted to bind the SIM3 peptide (\u003cstrong\u003eFigure 6E\u003c/strong\u003e). 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 (\u003cstrong\u003eFigure S8\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo evaluate the affinity of Smt3 for Mre11-SIM3, we performed isothermal titration calorimetry (ITC) binding experiments between the Mre11-SIM3 peptides and Smt3. ITC analysis confirmed that the wild-type SIM3 peptide binds Smt3 with a K\u003csub\u003eD\u0026nbsp;\u003c/sub\u003eof 20\u0026nbsp;±\u0026nbsp;10 µM, typical for SUMO-SIM interactions\u003csup\u003e49,50\u003c/sup\u003e. In contrast, the mutant SIM3 peptide failed to interact with Smt3 (\u003cstrong\u003eFigures 6F and S9\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSIM3 contributes to Mre11 recruitment and meiotic DSB formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the role of Mre11-SIM3 \u003cem\u003ein vivo\u003c/em\u003e, the core SIM3 motif (IIMVS) was substituted with alanines. \u003cem\u003emre11-SIM3\u003c/em\u003e mutant cells showed a small reduction in meiotic Mre11 foci (\u003cstrong\u003eFigure 7A\u003c/strong\u003e), which was associated with delayed and reduced DSB formation (\u003cstrong\u003eFigure 7B and S10C\u003c/strong\u003e). Combining the \u003cem\u003eSIM3\u003c/em\u003e mutation with truncation of the Mre11 C-terminal\u0026nbsp;a-helix (\u003cem\u003emre11-ΔC15+SIM3\u003c/em\u003e) further reduced Mre11 foci and abolished DSB formation (\u003cstrong\u003eFigures 7A and 7B\u003c/strong\u003e), while protein level remained unchanged (\u003cstrong\u003eFigures S10A and S10B\u003c/strong\u003e). Consequently, the \u003cem\u003emre11-SIM3\u0026nbsp;\u003c/em\u003emutant had slightly reduced spore viability (86%), while \u003cem\u003emre11-ΔC15+SIM3\u003c/em\u003e spores were completely inviable (\u003cstrong\u003eFigures 7C)\u003c/strong\u003e.\u0026nbsp;The \u003cem\u003emre11-SIM3\u0026nbsp;\u003c/em\u003emutant also showed a minor delay in meiotic progression; while progression was accelerated in the \u003cem\u003emre11-ΔC15+SIM3\u0026nbsp;\u003c/em\u003emutant compared to the wild-type, likely due to the absence of DNA breaks \u003cstrong\u003e(Figure S10D)\u003c/strong\u003e.\u0026nbsp;We conclude that SIM3 promotes the recruitment of Mre11 to the meiotic DSB machinery.\u003c/p\u003e\n\u003cp\u003eTo test the hypothesis that SIM3 contributes to the interaction between Mre11 and Mer2, we analyzed Mre11-Mer2 interaction using the yeast-two-hybrid assay. While truncation of the terminal 15 amino acids of Mre11 abolished the interaction with Mer2, mutation of the SIM3 motif had no discernable impact (\u003cstrong\u003eFigure S10E\u003c/strong\u003e). We note, however, that Mer2 may not be SUMOylated in this system, which employs mitotically cycling cells. Hence, the potential contribution of Mer2 SUMOylation to productive interaction with Mre11 during meiosis remains unclear.\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003eBudding yeast Mre11 has a C-terminal tail with meiosis-specific functions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MRX complex plays key functions in the maintenance of genomic integrity throughout eukaryotes\u003csup\u003e51,52\u003c/sup\u003e. However, its role in promoting the formation of Spo11-dependent DSBs during meiosis has only been reported in budding yeast and \u003cem\u003eC. elegans\u003c/em\u003e\u003csup\u003e15–17,53\u003c/sup\u003e. Indeed, MRX orthologs are not required for meiotic DSB formation in \u003cem\u003eA. thaliana\u003c/em\u003e\u003csup\u003e54\u003c/sup\u003e, \u003cem\u003eS. pombe\u003c/em\u003e\u003csup\u003e55\u003c/sup\u003e, and mice\u003csup\u003e56,57\u003c/sup\u003e. In \u003cem\u003eC. elegans\u003c/em\u003e, MRE-11 and RAD-50 are necessary for DSB formation\u003csup\u003e53,58\u003c/sup\u003e, but the ortholog of Xrs2, NBS-1, is not\u003csup\u003e59\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eS. cerevisiae\u003c/em\u003e, the binding of Mre11 to DSB hotspots depends on the presence of all other DSB proteins except Rad50\u003csup\u003e40\u003c/sup\u003e. This suggests that the MRX complex may be the final component recruited to the DSB machinery and raises the possibility that MRX recruitment may trigger Spo11’s catalytic activity, though the underlying mechanism remains unknown. It has been suggested that the requirement for the MRX complex prior to DSB formation serves to coordinate DSB formation with downstream repair, thereby minimizing genomic instability\u003csup\u003e40\u003c/sup\u003e. Supporting this idea, DSBs detected in wild-type cells are typically fully resected, indicating that processing occurs more rapidly than break accumulation. This observation is consistent with tight coordination between DSB formation and repair\u003csup\u003e60–63\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIt was previously shown that the C-terminus of Mre11 is dispensable for DSB repair in mitotically cycling cells, but essential for the formation of meiotic DSBs\u003csup\u003e29,40\u003c/sup\u003e. Here, we show that this essential meiotic function of the Mre11 C-terminus involves multiple mechanisms that collaborate to promote Mre11 recruitment to recombination sites (\u003cstrong\u003eFigure 7D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Mre11 C-terminus promotes meiotic DSB formation via multiple mechanisms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, we show that the C-terminal\u0026nbsp;a-helix of Mre11 directly binds the Mer2 SSM1 motif located on the N-terminal side of the tetrameric coiled coil. AlphaFold modeling and mutagenesis identified Mre11-LLK and Mer2-EQEK residues as being responsible for this interaction.\u0026nbsp;Truncating the Mre11 terminal a-helix or mutating the Mer2-EQEK residues \u003cem\u003ein vivo\u003c/em\u003e confirmed the functional importance of this interaction for Mre11 foci formation and DSB formation.\u0026nbsp;These data are in line with previous yeast-two-hybrid and pulldown experiments that revealed an interaction between Mre11 and Mer2\u003csup\u003e41\u003c/sup\u003e. We note, however, that the Mer2 alleles previously reported to reduce Mre11 interaction involved amino acids predicted to point inside the Mer2 tetrameric coiled coil, which are therefore likely to affect Mre11 interaction indirectly by compromising the structural stability of Mer2 (\u003cstrong\u003eFigure S5C\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSecond, we identified and biochemically validated a novel SUMO-interacting motif located within the Mre11 IDR and showed that this motif also participates in Mre11 recruitment and DSB formation during meiosis. While the meiotic phenotypes of the \u003cem\u003emre11-SIM3\u003c/em\u003e mutant were relatively modest, we find that SIM3 collaborates with the C-terminal\u0026nbsp;a-helix\u0026nbsp;of Mre11 for productive recruitment to precursor DSB sites.\u003c/p\u003e\n\u003cp\u003eThird, a recent study demonstrated that the Mre11 C-terminus is also important for direct interaction with Spo11\u003csup\u003e31\u003c/sup\u003e. While the binding sites were not precisely identified, this interaction implicates Mre11 residues 663 to 676, just upstream of the terminal\u0026nbsp;a-helix. It was noted that a mutant lacking the last 16 amino acids of Mre11 was defective in meiosis despite no effect on Spo11 binding, indicating that interaction with Spo11 is not sufficient for functional recruitment of Mre11. Our data explains this result by demonstrating that the Mre11 terminal\u0026nbsp;a-helix binds to Mer2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, we showed that the C-terminal region of Mre11 is also required to assemble DNA-dependent condensates \u003cem\u003ein vitro\u003c/em\u003e. DNA nucleates Mre11 condensates and acts as a scaffold that then recruits free soluble protein through homotypic self-association. We show that the C-terminal 49 residues of Mre11 are sufficient for self-association when Mer2 condensates are provided as a nucleation site, suggesting that this may constitute yet another meiosis-specific function of the Mre11 C-terminal tail. Nevertheless, in the absence of a mutant that specifically abolishes Mre11 self-association, the functional importance of this activity for meiotic DSB formation remains unclear.\u003c/p\u003e\n\u003cp\u003eIn summary, the recruitment of Mre11 during meiosis involves at least three, perhaps four, independent functions of the Mre11 C-terminal tail.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssembly of the DSB machinery by hierarchical condensation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e condensation activity of Mre11 and MRX are reminiscent of that of Rec114-Mei4 and Mer2 that also form dynamic and reversible macromolecular assemblies in the presence of DNA\u003csup\u003e9\u003c/sup\u003e. Similar to RMM, Mre11 and MRX condensates likely involve electrostatic interactions between the negatively charged DNA backbone and positively charged residues present in the C-terminal region of Mre11. Condensates are further stabilized by the presence of positively charged ions such as magnesium that presumably inhibit intramolecular repulsion within the DNA substrate. In addition, sensitivity of Mre11 condensates to 1,6-hexanediol indicates that self-association via the low-complexity IDR depends on weak hydrophobic interactions, further supporting the liquid nature of these assemblies.\u003c/p\u003e\n\u003cp\u003eWhile both Mre11 and MRX condensates share similar properties, they don’t fully mirror each other in their behavior, suggesting that the presence of Rad50 and Xrs2 might confer additional stabilization to MRX condensates, consistent with a previous observation that Rad50 self-interaction drives Mre11-Rad50 oligomerization\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe spontaneously assembly of Mer2, Rec114-Mei4 and Mre11/MRX condensates \u003cem\u003ein vitro\u003c/em\u003e contrasts with the more stringent assembly of their respective foci \u003cem\u003ein vivo\u003c/em\u003e. Indeed, Mer2 foci depend on the axis protein Hop1, Rec114 foci depend on Mer2, and Mre11 foci depend on Mer2 and likely most other DSB proteins\u003csup\u003e14,40,64–66\u003c/sup\u003e. We propose that the DSB machinery assembles via a hierarchical mechanism of successive condensate nucleation and growth events, where Hop1 nucleates the assembly of Mer2 condensates that nucleate Rec114-Mei4 condensates that recruit the Spo11 complex\u003csup\u003e9\u003c/sup\u003e. Mer2 condensates also recruit Mre11 and drive its self-assembly, dependent on stabilizing interactions with Spo11 and other SUMOylated targets (\u003cstrong\u003eFigure 7D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe importance of a SUMO-SIM interaction in Mre11 recruitment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIf the recruitment of Mre11 constitutes the final step in the assembly of the DSB machinery prior to triggering Spo11-dependent cleavage, SUMOylation of the DSB machinery could serve to mark licensed pre-DSB complexes and/or allow for reversible interactions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhat are the SUMOylated targets bound by Mre11 prior to DSB formation? SUMOylation was previously shown to regulate the key events of meiotic prophase I, including DSB formation, and thousands of SUMOylation sites were mapped on meiotic proteins\u003csup\u003e45\u003c/sup\u003e. Amongst those, thirteen sites were identified within Mer2, including several close to the SSM1 motif. Given the physical proximity of the Mre11\u003csup\u003eLLK\u0026nbsp;\u003c/sup\u003eand Mer2\u003csup\u003eEQEK\u003c/sup\u003e interaction regions and Mre11-SIM3, it is tempting to speculate that SIM3 might be interacting with SUMOylated Mer2 during meiosis, which would presumably serve as an anchor to stabilize the interaction with Mer2. However, the recruitment of Mre11 through the SIM3 motif may involve other SUMOylated DSB proteins, including Spp1, Rec114, Hop1, Red1, and cohesin\u003csup\u003e45\u003c/sup\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003ePreparation of expression vectors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOligonucleotides (oligos) used in this study were purchased from Sigma-Aldrich. The sequences of the oligos used are listed in \u003cstrong\u003eTable S1\u003c/strong\u003e. Plasmids generated in this study were verified by sequencing and are listed in \u003cstrong\u003eTable S2\u003c/strong\u003e. Peptides used were ordered from GenScript or synthesized in the Ballet laboratory and are listed in \u003cstrong\u003eTable S3\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe expression vector for Mre11\u003csup\u003e10xHis\u003c/sup\u003e was produced by PCR amplification of the \u003cem\u003eMRE11\u003c/em\u003e 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 \u003csup\u003eeGFP\u003c/sup\u003eMre11, pCCB942. The expression vector for Rad50 was produced by PCR amplification of the \u003cem\u003eRAD50\u003c/em\u003e 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 Xrs2\u003csup\u003e2xFlag\u003c/sup\u003e was produced by PCR amplification of the \u003cem\u003eXRS2\u003c/em\u003e 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExpression vectors for \u003csup\u003eeGFP\u003c/sup\u003eMre11\u003csup\u003e10xHis\u003c/sup\u003e-\u003cem\u003eΔ10-270\u003c/em\u003e (pry7), \u003csup\u003eeGFP\u003c/sup\u003eMre1\u003csup\u003e10xHis\u003c/sup\u003e-\u003cem\u003eΔ290-472\u003c/em\u003e (pry5), \u003csup\u003eeGFP\u003c/sup\u003eMre1\u003csup\u003e10xHis\u003c/sup\u003e-Δ\u003cem\u003eIDR\u003c/em\u003e (pry6), and \u003csup\u003eeGFP\u003c/sup\u003eMre1\u003csup\u003e10xHis\u003c/sup\u003e-Δ\u003cem\u003eC49\u003c/em\u003e (pCCB943) were amplified by inverse PCR from pCCB942 using primers pp26 and pp27, pp28 and pp29, pp24 and pp25, and cb1435 and cb1458, respectively. The amplified product was gel extracted, phosphorylated, and ligated to generate the truncations. The expression vector for \u003csup\u003eeGFP\u003c/sup\u003eMre11-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.\u003c/p\u003e\n\u003cp\u003eThe vector for expression of \u003csup\u003eMBP\u003c/sup\u003eMre11-C49 and \u003csup\u003eHisSUMO\u003c/sup\u003eMer2 (pCCB1040) was based on a pET-Duet1 vector with the sequence coding for \u003csup\u003eMBP\u003c/sup\u003eMre11-C49 cloned within the first position (SacI site) and the sequence coding for \u003csup\u003eHisSUMO\u003c/sup\u003eMer2 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe expression vector for \u003csup\u003emScarlet\u003c/sup\u003eMre11-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. Expression vectors for \u003csup\u003eAlexa594\u003c/sup\u003eMer2 (pCCB750) and \u003csup\u003eeGFP\u003c/sup\u003eMer2 (pCCB777) were previously described\u003csup\u003e9\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pET28b expression vector for Smt3 (pCCB998) was a kind gift from Chris Lima\u003csup\u003e67\u003c/sup\u003e. 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-\u003cem\u003eSIM3\u003c/em\u003e (pNH1371)\u0026nbsp;and RB268 and RB269 to generate\u0026nbsp;pGAD-C1-Mre11-\u003cem\u003eΔC15\u003c/em\u003e (pNH1372).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression and purification of recombinant proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecombinant baculoviruses were produced by Bac-to-Bac Baculovirus Expression System (Invitrogen) following the manufacturer’s instructions. For every induction, 1 L culture containing 2 × 10\u003csup\u003e6\u003c/sup\u003e \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e (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 Mre11\u003csup\u003e10xHis\u003c/sup\u003e-Rad50-Xrs2\u003csup\u003e2xFLAG\u003c/sup\u003e (MRX) and pCCB942, pCCB866, and pCCB867 were used for the expression of \u003csup\u003eeGFP\u003c/sup\u003eMre11\u003csup\u003e10xHis\u003c/sup\u003e-Rad50-Xrs2\u003csup\u003e2xFLAG\u003c/sup\u003e (\u003csup\u003eeGFP\u003c/sup\u003eMRX). Full-length Mre11\u003csup\u003e10xHis\u003c/sup\u003e and \u003csup\u003eeGFP\u003c/sup\u003eMre11\u003csup\u003e10xHis\u003c/sup\u003e were expressed using viruses generated from pCCB856 and pCCB942, respectively. Mre11 truncations \u003csup\u003eeGFP\u003c/sup\u003eMre11\u003csup\u003e10xHis\u003c/sup\u003e-\u003cem\u003eΔ10-270\u003c/em\u003e, \u003csup\u003eeGFP\u003c/sup\u003eMre11\u003csup\u003e10xHis\u003c/sup\u003e-\u003cem\u003eΔ290-472\u003c/em\u003e, \u003csup\u003eeGFP\u003c/sup\u003eMre11\u003csup\u003e10xHis\u003c/sup\u003e-\u003cem\u003eΔIDR\u003c/em\u003e, and \u003csup\u003eeGFP\u003c/sup\u003eMre11\u003csup\u003e10xHis\u003c/sup\u003e-\u003cem\u003eΔC49\u003c/em\u003e were expressed using viruses generated from pry7, pry5, pry6, and pCCB943, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrior 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe following protocol was used for the purification of His-tagged \u003csup\u003eeGFP\u003c/sup\u003eMre11 and \u003csup\u003eeGFP\u003c/sup\u003eMre11 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.\u003c/p\u003e\n\u003cp\u003eFor 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.\u003c/p\u003e\n\u003cp\u003ePurification of recombinant His- and Flag-tagged \u003csup\u003eeGFP\u003c/sup\u003eMRX complexes and truncations were performed essentially as described\u003csup\u003e68\u003c/sup\u003e. 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor expression of recombinant \u003csup\u003emScarlet\u003c/sup\u003eMre11-C49 in \u003cem\u003eE. coli\u003c/em\u003e, pry109 was 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 (OD\u003csub\u003e600\u003c/sub\u003e) of 0.6. Expression was carried out for 20 hours at 16°C with 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) 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. 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). 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExpression and purification of \u003csup\u003eeGFP\u003c/sup\u003eMer2 and \u003csup\u003eAlexa594\u003c/sup\u003eMer2 were performed as previously described\u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor expression of recombinant Smt3\u003csup\u003e6xHis\u003c/sup\u003e in \u003cem\u003eE. coli\u003c/em\u003e, 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 OD\u003csub\u003e600\u003c/sub\u003e 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 20,000 rpm 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the production of the doubly labeled U-[\u003csup\u003e13\u003c/sup\u003eC, \u003csup\u003e15\u003c/sup\u003eN] Smt3\u003csup\u003e6xHis\u003c/sup\u003e protein, the IPTG induction was carried out in minimal medium containing M9 salts (6.8 g/L Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 3 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and 1 g/L NaCl), 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, trace elements (60 mg/L FeSO\u003csub\u003e4\u003c/sub\u003e·7H\u003csub\u003e2\u003c/sub\u003eO, 12 mg/L MnCl\u003csub\u003e2\u003c/sub\u003e·4H\u003csub\u003e2\u003c/sub\u003eO, 8 mg/L CoCl\u003csub\u003e2\u003c/sub\u003e·6H\u003csub\u003e2\u003c/sub\u003eO, 7 mg/L ZnSO\u003csub\u003e4\u003c/sub\u003e·7H\u003csub\u003e2\u003c/sub\u003eO, 3 mg/L CuCl\u003csub\u003e2\u003c/sub\u003e·2H\u003csub\u003e2\u003c/sub\u003eO, 0.2 mg/L H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, and 50 mg/L EDTA), BME vitamin mix (Sigma), and 1 g/L \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl and 2 g/L [\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e]glucose (CortecNet) as the sole nitrogen and carbon sources, respectively. The purification protocol remained the same as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;condensation assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins 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 MgCl\u003csub\u003e2\u003c/sub\u003e, 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 \u003csup\u003eAlexa488\u003c/sup\u003eMre11 or 100 nM \u003csup\u003eeGFP\u003c/sup\u003eMRX. 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 mins, 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 script\u003csup\u003e9\u003c/sup\u003e. 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGel shift assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast targeting vectors and strain construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYeast strains are generated from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e SK1 background and are listed in \u003cstrong\u003eTable 4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo produce a yeast targeting vector, \u003cem\u003eMRE11-myc8::URA3\u003c/em\u003e 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 \u003cem\u003emre11-ΔIDR\u003c/em\u003e (pry30), \u003cem\u003emre11-ΔC49\u003c/em\u003e (pry24), and \u003cem\u003emre11-ΔC15\u003c/em\u003e (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 \u003cem\u003emre11-\u003c/em\u003e\u003cem\u003eSIM\u003c/em\u003e (pry56) and \u003cem\u003emre11-SIM+ΔC15\u003c/em\u003e (pry57) were generated similarly using primers pp3 and pp4 and templates pry2 and pry42, respectively. Genomic integration of wild-type and truncated \u003cem\u003eMRE11-myc8::URA3\u003c/em\u003e cassettes was performed by SpeI and NotI digestion of the corresponding plasmids and insertion in the endogenous \u003cem\u003eMRE11\u003c/em\u003e locus of strain CBY006 by ‘LiAc’-based transformation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlasmids to produce \u003cem\u003eMER2\u003c/em\u003e mutant strains were based on pMH002, which contains a \u003cem\u003eMER2::HphMX\u003c/em\u003e cassette cloned into a TOPO-based vector, as described\u003csup\u003e43\u003c/sup\u003e. To generate \u003cem\u003emer2-EQEK\u003c/em\u003e, 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 (Mer2\u003csup\u003eiV5\u003c/sup\u003e) using primers dam005 and dam006 on plasmid backbone pMH002 by inverse PCR followed by ligation to generate pDAM003. \u003cem\u003emer2\u003csup\u003eiV5\u003c/sup\u003e-EQEK\u003c/em\u003e was constructed by inverse PCR followed by ligation of pDAM003 using primers cb1451 and cb1562. Genomic integration of \u003cem\u003emer2-EQEK::HphMX\u0026nbsp;\u003c/em\u003eand \u003cem\u003emer2\u003csup\u003eiV5\u003c/sup\u003e-EQEK::HphMX\u003c/em\u003e cassettes was performed by SpeI, NotI, and XmaI digestion of the respective plasmids and insertion into the endogenous \u003cem\u003eMER2\u003c/em\u003e locus of CBY006 by ‘LiAc’-based transformation. The \u003cem\u003eMER2\u003csup\u003eiV5\u003c/sup\u003e::HphMX\u003c/em\u003e allele was constructed similarly following BamHI and SphI digestion of pDAM003.\u003c/p\u003e\n\u003cp\u003eAll 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 \u003cstrong\u003eTable S4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpore viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast culture and meiotic synchronization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing 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 OD\u003csub\u003e600\u003c/sub\u003e 0.2 and grown for 12-14 hours at 30°C, 250 rpm. Once the cultures reached OD\u003csub\u003e600\u003c/sub\u003e 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 OD\u003csub\u003e600\u003c/sub\u003e 0.2 and grown for 3-4 hours to reach OD\u003csub\u003e600\u003c/sub\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpreading and immunofluorescence of yeast nuclei spreads\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMeiotic 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 MES-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.\u003c/p\u003e\n\u003cp\u003eSlides 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 H2A-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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting of yeast meiotic extracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMeiotic cultures at desired timepoints were harvested, washed in ice-cold water, and lysed in 20% trichloroacetic acid (TCA) by agitation in a bead beater (insert company) using 0.5 mm zirconia/silica beads (insert company). 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSouthern Blotting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMeiotic DSB analysis by Southern blotting was performed as previously described\u003csup\u003e69\u003c/sup\u003e. In brief, synchronized cultures undergoing meiosis were collected at the indicated time points. After DNA isolation, 1\u0026nbsp;µg of genomic DNA was digested by\u0026nbsp;PstI\u0026nbsp;and separated on a 1% TBE-agarose gel. DNA was transferred to Amersham™ Hybond™-N+ nylon membranes (Cytiva) by vacuum transfer, hybridized with \u003cem\u003eGAT1\u003c/em\u003e probe (amplified with primers: 5′-CGCGCTTCACATAATGCTTCTGG, 5'-TTCAGATTCAACCAATCCAGGCTC) and developed by autoradiography.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePull-down assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWild-type and mutant MBP-tagged Mre11-C49 and HisSUMO-tagged Mer2 were co-expressed in 50 mL of \u003cem\u003eE. coli\u003c/em\u003e 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNuclear magnetic resonance spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples contained 0.3-0.6 mM of U-[\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e15\u003c/sup\u003eN] Smt3 in 20 mM sodium phosphate, 20 mM NaCl (pH 6.5), 0.02% NaN\u003csub\u003e3\u003c/sub\u003e and 10% D\u003csub\u003e2\u003c/sub\u003eO 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 processed in TopSpin 3.6 (Bruker) or NMRPipe\u003csup\u003e70\u003c/sup\u003e and analyzed in CCPNMR\u003csup\u003e71\u003c/sup\u003e. Assignments of Smt3 backbone amide resonances were taken from literature\u003csup\u003e72\u003c/sup\u003e and verified by 3D HNCACB, HN(CO)CACB, and \u003csup\u003e15\u003c/sup\u003eN-edited NOESY-HSQC spectra. Further assignments of methyl resonances were obtained from 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 \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e15\u003c/sup\u003eN 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe 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-[\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e15\u003c/sup\u003eN] Smt3, with the spectral changes monitored in [\u003csup\u003e1\u003c/sup\u003eH,\u003csup\u003e15\u003c/sup\u003eN] HSQC spectra acquired at each increment. The average chemical shift perturbations (Δδ\u003csub\u003eavg\u003c/sub\u003e) were calculated as Δδ\u003csub\u003eavg\u003c/sub\u003e = (Δδ\u003csub\u003eX\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e/50 + Δδ\u003csub\u003eH\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e/2)\u003csup\u003e0.5\u003c/sup\u003e, where Δδ\u003csub\u003eX\u003c/sub\u003e and Δδ\u003csub\u003eH\u003c/sub\u003e are the chemical shift changes of the backbone amide nitrogens or methyl carbons (Δδ\u003csub\u003eX\u003c/sub\u003e) and protons (Δδ\u003csub\u003eH\u003c/sub\u003e) 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeptide synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe peptides were synthesized by standard Fmoc-based solid phase peptide synthesis. Preloaded Fmoc-Glu-Wang resin (0.55 mmol/g) was swollen in \u003cem\u003eN,N\u003c/em\u003e-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 \u003cem\u003eN,N\u003c/em\u003e-diisopropylethylamine (6 equiv.). After synthesis of the full sequence on resin, the peptide was cleaved with trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H\u003csub\u003e2\u003c/sub\u003eO (90/5/5, \u003cem\u003ev/v\u003c/em\u003e). The products were purified by preparative reverse phase HPLC using an acetonitrile/water eluent mixture containing 0.1% TFA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsothermal titration calorimetry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast two-hybrid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBait 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis and data visualization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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 \u003cem\u003eP\u003c/em\u003e-value calculation (p ≥ 0.05, ns, not significant; **\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt;\u0026nbsp;0.01; ***\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt;\u0026nbsp;0.001; ****\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank David Alvarez Melo for generating Mer2-iV5 tagged strains, John Weir for plasmids and strains, and CCB laboratory members for discussion. We also thank Biological Imaging facility (IMABIOL) at UCLouvain and Marie-Christine Eloy for providing training in the use of the epifluorescence microscope. This work was supported by the European Research Council under the European Union\u0026rsquo;s Horizon 2020 research and innovation program (ERC grant agreement 802525 to CCB), and the Fonds National de la Recherche Scientifique (PDR grant T.0031.22 to CCB). PP is funded by FNRS Aspirant fellowships (project 1.A908.22). CCB is a FNRS Research Associate. WEYM and SB acknowledge the Research Council of VUB for support through the Strategic Research Program SRP95 and the infrastructure grant OZR3939. NIH NIGMS grant R01GM074223 supported NH, who is also an Investigator of the Howard Hughes Medical Institute.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.P. designed, executed, and analyzed all experiments except as noted; M. S. performed Southern blot experiments; W.E.Y.M. synthesized peptides under the supervision of S.B., performed ITC experiments and analyzed NMR data; A.N.V. acquired and analyzed NMR data; R.B. performed yeast 2-hybrid experiments under the supervision of N.H.;\u0026nbsp;C.C.B. supervised the research and secured funding. P.P. and C.C.B wrote the paper with input from all authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA damage: mechanisms, mutation, and disease. \u003cem\u003eFASEB j\u003c/em\u003e. 2003;17(10):1195-1214. doi:10.1096/fj.02-0752rev\u003c/li\u003e\n\u003cli\u003eDe Almeida LC, Calil FA, Machado-Neto JA, Costa-Lotufo LV. DNA damaging agents and DNA repair: From carcinogenesis to cancer therapy. \u003cem\u003eCancer Genetics\u003c/em\u003e. 2021;252-253:6-24. doi:10.1016/j.cancergen.2020.12.002\u003c/li\u003e\n\u003cli\u003eTubbs A, Nussenzweig A. Endogenous DNA Damage as a Source of Genomic Instability in Cancer. \u003cem\u003eCell\u003c/em\u003e. 2017;168(4):644-656. doi:10.1016/j.cell.2017.01.002\u003c/li\u003e\n\u003cli\u003eOster S, Aqeilan RI. Programmed DNA Damage and Physiological DSBs: Mapping, Biological Significance and Perturbations in Disease States. \u003cem\u003eCells\u003c/em\u003e. 2020;9(8):1870. doi:10.3390/cells9081870\u003c/li\u003e\n\u003cli\u003eB\u0026ouml;rner GV, Hochwagen A, MacQueen AJ. Meiosis in budding yeast. \u003cem\u003eGENETICS\u003c/em\u003e. 2023;225(2):iyad125. doi:10.1093/genetics/iyad125\u003c/li\u003e\n\u003cli\u003eZickler D, Kleckner N. Meiosis: Dances Between Homologs. \u003cem\u003eAnnu Rev Genet\u003c/em\u003e. 2023;57(1):1-63. doi:10.1146/annurev-genet-061323-044915\u003c/li\u003e\n\u003cli\u003eLam I, Keeney S. Mechanism and Regulation of Meiotic Recombination Initiation. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e. 2015;7(1):a016634. doi:10.1101/cshperspect.a016634\u003c/li\u003e\n\u003cli\u003eYadav VK, Claeys Bouuaert C. Mechanism and Control of Meiotic DNA Double-Strand Break Formation in S. cerevisiae. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e. 2021;9:642737. doi:10.3389/fcell.2021.642737\u003c/li\u003e\n\u003cli\u003eClaeys Bouuaert C, Pu S, Wang J, et al. DNA-driven condensation assembles the meiotic DNA break machinery. \u003cem\u003eNature\u003c/em\u003e. 2021;592(7852):144-149. doi:10.1038/s41586-021-03374-w\u003c/li\u003e\n\u003cli\u003eClaeys Bouuaert C, Tischfield SE, Pu S, et al. Structural and functional characterization of the Spo11 core complex. \u003cem\u003eNat Struct Mol Biol\u003c/em\u003e. 2021;28(1):92-102. doi:10.1038/s41594-020-00534-w\u003c/li\u003e\n\u003cli\u003eJohnson D, Crawford M, Cooper T, et al. Concerted cutting by Spo11 illuminates meiotic DNA break mechanics. \u003cem\u003eNature\u003c/em\u003e. 2021;594(7864):572-576. doi:10.1038/s41586-021-03389-3\u003c/li\u003e\n\u003cli\u003eOger C, Claeys Bouuaert C. SPO11 dimers are sufficient to catalyse DNA double-strand breaks in vitro. \u003cem\u003eNature\u003c/em\u003e. 2025;639(8055):792-799. doi:10.1038/s41586-024-08574-8\u003c/li\u003e\n\u003cli\u003eKleckner N. Chiasma formation: chromatin/axis interplay and the role(s) of the synaptonemal complex. \u003cem\u003eChromosoma\u003c/em\u003e. 2006;115(3):175-194. doi:10.1007/s00412-006-0055-7\u003c/li\u003e\n\u003cli\u003ePanizza S, Mendoza MA, Berlinger M, et al. Spo11-Accessory Proteins Link Double-Strand Break Sites to the Chromosome Axis in Early Meiotic Recombination. \u003cem\u003eCell\u003c/em\u003e. 2011;146(3):372-383. doi:10.1016/j.cell.2011.07.003\u003c/li\u003e\n\u003cli\u003eAlani E, Padmore R, Kleckner N. Analysis of Wild-Type and rad50 mutants of Yeast Suggests an Intimate Relationship between Meiotic Chromosome Synapsis and Recombination. \u003cem\u003eCell\u003c/em\u003e. 1990;61(3):419-436. doi:10.1016/0092-8674(90)90524-I\u003c/li\u003e\n\u003cli\u003eIvanov EL, Korolevt VG, Fabre F. Xrs2, a DNA Repair Gene of Saccharomyces Cerevisiae, Is Needed for Meiotic Recombination. \u003cem\u003eGENETICS\u003c/em\u003e. 1992;132(3):651-664. doi:10.1093/genetics/132.3.651\u003c/li\u003e\n\u003cli\u003eNairz K, Klein F. \u003cem\u003emre11S\u003c/em\u003e \u0026mdash;a yeast mutation that blocks double-strand-break processing and permits nonhomologous synapsis in meiosis. \u003cem\u003eGenes Dev\u003c/em\u003e. 1997;11(17):2272-2290. doi:10.1101/gad.11.17.2272\u003c/li\u003e\n\u003cli\u003eKeeney S. Mechanism and control of meiotic recombination initiation. \u003cem\u003eCurrent Topics in Developmental Biology\u003c/em\u003e. 2001;52:1-53. doi:10.1016/S0070-2153(01)52008-6\u003c/li\u003e\n\u003cli\u003eMoore JK, Haber JE. Cell Cycle and Genetic Requirements of Two Pathways of Nonhomologous End-Joining Repair of Double-Strand Breaks in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. \u003cem\u003eMolecular and Cellular Biology\u003c/em\u003e. 1996;16(5):2164-2173. doi:10.1128/MCB.16.5.2164\u003c/li\u003e\n\u003cli\u003ePaull TT, Gellert M. The 3\u0026prime; to 5\u0026prime; Exonuclease Activity of Mre11 Facilitates Repair of DNA Double-Strand Breaks. \u003cem\u003eMolecular Cell\u003c/em\u003e. 1998;1(7):969-979. doi:10.1016/S1097-2765(00)80097-0\u003c/li\u003e\n\u003cli\u003eGarcia V, Phelps SEL, Gray S, Neale MJ. Bidirectional resection of DNA double-strand breaks by Mre11 and Exo1. \u003cem\u003eNature\u003c/em\u003e. 2011;479(7372):241-244. doi:10.1038/nature10515\u003c/li\u003e\n\u003cli\u003eCasari E, Rinaldi C, Marsella A, et al. Processing of DNA Double-Strand Breaks by the MRX Complex in a Chromatin Context. \u003cem\u003eFront Mol Biosci\u003c/em\u003e. 2019;6:43. doi:10.3389/fmolb.2019.00043\u003c/li\u003e\n\u003cli\u003eGobbini E, Cassani C, Villa M, Bonetti D, Longhese M. Functions and regulation of the MRX complex at DNA double-strand breaks. \u003cem\u003eMicrobial Cell\u003c/em\u003e. 2016;3(8):329-337. doi:10.15698/mic2016.08.517\u003c/li\u003e\n\u003cli\u003ePaull TT. 20 Years of Mre11 Biology: No End in Sight. \u003cem\u003eMolecular Cell\u003c/em\u003e. 2018;71(3):419-427. doi:10.1016/j.molcel.2018.06.033\u003c/li\u003e\n\u003cli\u003eTisi R, Vertemara J, Zampella G, Longhese MP. Functional and structural insights into the MRX/MRN complex, a key player in recognition and repair of DNA double-strand breaks. \u003cem\u003eComputational and Structural Biotechnology Journal\u003c/em\u003e. 2020;18:1137-1152. doi:10.1016/j.csbj.2020.05.013\u003c/li\u003e\n\u003cli\u003eLamarche BJ, Orazio NI, Weitzman MD. The MRN complex in double‐strand break repair and telomere maintenance. \u003cem\u003eFEBS Letters\u003c/em\u003e. 2010;584(17):3682-3695. doi:10.1016/j.febslet.2010.07.029\u003c/li\u003e\n\u003cli\u003eLu R, Zhang H, Jiang YN, Wang ZQ, Sun L, Zhou ZW. Post-Translational Modification of MRE11: Its Implication in DDR and Diseases. \u003cem\u003eGenes\u003c/em\u003e. 2021;12(8):1158. doi:10.3390/genes12081158\u003c/li\u003e\n\u003cli\u003eOh J, Symington LS. Role of the Mre11 Complex in Preserving Genome Integrity. \u003cem\u003eGenes\u003c/em\u003e. 2018;9(12):589. doi:10.3390/genes9120589\u003c/li\u003e\n\u003cli\u003eFuruse M, Nagase Y, Tsubouchi H, Murakami-Murofushi K, Shibata T, Ohta K. Distinct roles of two separable \u003cem\u003ein vitro\u003c/em\u003e activities of yeast Mre11 in mitotic and meiotic recombination. \u003cem\u003eThe EMBO Journal\u003c/em\u003e. 1998;17(21):6412-6425. doi:10.1093/emboj/17.21.6412\u003c/li\u003e\n\u003cli\u003eUsui T, Ohta T, Oshiumi H, Tomizawa J ichi, Ogawa H, Ogawa T. Complex Formation and Functional Versatility of Mre11 of Budding Yeast in Recombination. \u003cem\u003eCell\u003c/em\u003e. 1998;95(5):705-716. doi:10.1016/S0092-8674(00)81640-2\u003c/li\u003e\n\u003cli\u003eAithal R, Nangalia K, Spirek M, Chen D, Klein F, Krejci L. Physical interaction with Spo11 mediates the localisation of Mre11 to chromatin in meiosis and promotes its nuclease activity. \u003cem\u003eNucleic Acids Research\u003c/em\u003e. 2024;52(8):4328-4343. doi:10.1093/nar/gkae111\u003c/li\u003e\n\u003cli\u003eWen Q, Scorah J, Phear G, Rodgers G, Rodgers S, Meuth M. A Mutant Allele of \u003cem\u003eMRE11\u003c/em\u003e Found in Mismatch Repair-deficient Tumor Cells Suppresses the Cellular Response to DNA Replication Fork Stress in a Dominant Negative Manner. Cohen-Fix O, ed. \u003cem\u003eMBoC\u003c/em\u003e. 2008;19(4):1693-1705. doi:10.1091/mbc.e07-09-0975\u003c/li\u003e\n\u003cli\u003eTamai T, Reginato G, Ojiri R, et al. Sae2 controls Mre11 endo- and exonuclease activities by different mechanisms. \u003cem\u003eNat Commun\u003c/em\u003e. 2024;15(1):7221. doi:10.1038/s41467-024-51493-5\u003c/li\u003e\n\u003cli\u003eKissling VM, Reginato G, Bianco E, et al. Mre11-Rad50 oligomerization promotes DNA double-strand break repair. \u003cem\u003eNat Commun\u003c/em\u003e. 2022;13(1):2374. doi:10.1038/s41467-022-29841-0\u003c/li\u003e\n\u003cli\u003eKroschwald S, Maharana S, Simon A. Hexanediol: a chemical probe to investigate the material properties of membrane-less compartments. \u003cem\u003eMatters\u003c/em\u003e. Published online May 22, 2017. doi:10.19185/matters.201702000010\u003c/li\u003e\n\u003cli\u003eLammens K, Bemeleit DJ, M\u0026ouml;ckel C, et al. The Mre11:Rad50 Structure Shows an ATP-Dependent Molecular Clamp in DNA Double-Strand Break Repair. \u003cem\u003eCell\u003c/em\u003e. 2011;145(1):54-66. doi:10.1016/j.cell.2011.02.038\u003c/li\u003e\n\u003cli\u003eRotheneder M, Stakyte K, Van De Logt E, et al. Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions. \u003cem\u003eMolecular Cell\u003c/em\u003e. 2023;83(2):167-185.e9. doi:10.1016/j.molcel.2022.12.003\u003c/li\u003e\n\u003cli\u003ePetrini J, Hohl M, Yu Y, Kuryavyi V, Patel D. Structure guided functional analysis of the S. cerevisiae Mre11 complex. Published online December 9, 2024. doi:10.21203/rs.3.rs-5390974/v1\u003c/li\u003e\n\u003cli\u003eBergerat A, De Massy B, Gadelle D, Varoutas PC, Nicolas A, Forterre P. An atypical topoisomerase II from archaea with implications for meiotic recombination. \u003cem\u003eNature\u003c/em\u003e. 1997;386(6623):414-417. doi:10.1038/386414a0\u003c/li\u003e\n\u003cli\u003eBorde V, Lin W, Novikov E, Petrini JH, Lichten M, Nicolas A. Association of Mre11p with Double-Strand Break Sites during Yeast Meiosis. \u003cem\u003eMolecular Cell\u003c/em\u003e. 2004;13(3):389-401. doi:10.1016/S1097-2765(04)00034-6\u003c/li\u003e\n\u003cli\u003eRousov\u0026aacute; D, Nivsarkar V, Altmannova V, et al. Novel mechanistic insights into the role of Mer2 as the keystone of meiotic DNA break formation. \u003cem\u003eeLife\u003c/em\u003e. 2021;10:e72330. doi:10.7554/eLife.72330\u003c/li\u003e\n\u003cli\u003eJumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. \u003cem\u003eNature\u003c/em\u003e. 2021;596(7873):583-589. doi:10.1038/s41586-021-03819-2\u003c/li\u003e\n\u003cli\u003eDaccache D, De Jonge E, Liloku P, et al. Evolutionary conservation of the structure and function of meiotic Rec114\u0026minus;Mei4 and Mer2 complexes. \u003cem\u003eGenes Dev\u003c/em\u003e. 2023;37(11-12):535-553. doi:10.1101/gad.350462.123\u003c/li\u003e\n\u003cli\u003eTess\u0026eacute; S, Bourbon HM, Debuchy R, et al. Asy2/Mer2: an evolutionarily conserved mediator of meiotic recombination, pairing, and global chromosome compaction. \u003cem\u003eGenes Dev\u003c/em\u003e. 2017;31(18):1880-1893. doi:10.1101/gad.304543.117\u003c/li\u003e\n\u003cli\u003eBhagwat NR, Owens SN, Ito M, et al. SUMO is a pervasive regulator of meiosis. \u003cem\u003eeLife\u003c/em\u003e. 2021;10:e57720. doi:10.7554/eLife.57720\u003c/li\u003e\n\u003cli\u003eChen YJ, Chuang YC, Chuang CN, et al. \u003cem\u003eS. cerevisiae\u003c/em\u003e Mre11 recruits conjugated SUMO moieties to facilitate the assembly and function of the Mre11-Rad50-Xrs2 complex. \u003cem\u003eNucleic Acids Res\u003c/em\u003e. 2016;44(5):2199-2213. doi:10.1093/nar/gkv1523\u003c/li\u003e\n\u003cli\u003eZhao Q, Xie Y, Zheng Y, et al. GPS-SUMO: a tool for the prediction of sumoylation sites and SUMO-interaction motifs. \u003cem\u003eNucleic Acids Research\u003c/em\u003e. 2014;42(W1):W325-W330. doi:10.1093/nar/gku383\u003c/li\u003e\n\u003cli\u003eLascorz J, Codina-Fabra J, Reverter D, Torres-Rosell J. SUMO-SIM interactions: From structure to biological functions. \u003cem\u003eSeminars in Cell \u0026amp; Developmental Biology\u003c/em\u003e. 2022;132:193-202. doi:10.1016/j.semcdb.2021.11.007\u003c/li\u003e\n\u003cli\u003eSong J, Zhang Z, Hu W, Chen Y. Small Ubiquitin-like Modifier (SUMO) Recognition of a SUMO Binding Motif. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e. 2005;280(48):40122-40129. doi:10.1074/jbc.M507059200\u003c/li\u003e\n\u003cli\u003eKeusekotten K, Bade VN, Meyer-Teschendorf K, et al. Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO. \u003cem\u003eBiochemical Journal\u003c/em\u003e. 2014;457(1):207-214. doi:10.1042/BJ20130753\u003c/li\u003e\n\u003cli\u003eSymington LS. Mechanism and regulation of DNA end resection in eukaryotes. \u003cem\u003eCritical Reviews in Biochemistry and Molecular Biology\u003c/em\u003e. 2016;51(3):195-212. doi:10.3109/10409238.2016.1172552\u003c/li\u003e\n\u003cli\u003eGn\u0026uuml;gge R, Symington LS. Keeping it real: MRX\u0026ndash;Sae2 clipping of natural substrates. \u003cem\u003eGenes Dev\u003c/em\u003e. 2017;31(23-24):2311-2312. doi:10.1101/gad.310771.117\u003c/li\u003e\n\u003cli\u003eChin GM, Villeneuve AM. \u003cem\u003eC. elegans mre-11\u003c/em\u003e is required for meiotic recombination and DNA repair but is dispensable for the meiotic G\u003csub\u003e2\u003c/sub\u003e DNA damage checkpoint. \u003cem\u003eGenes Dev\u003c/em\u003e. 2001;15(5):522-534. doi:10.1101/gad.864101\u003c/li\u003e\n\u003cli\u003ePuizina J, Siroky J, Mokros P, Schweizer D, Riha K. Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis. \u003cem\u003ePlant Cell\u003c/em\u003e. 2004;16(8):1968-1978. doi:10.1105/tpc.104.022749\u003c/li\u003e\n\u003cli\u003eYoung JA, Hyppa RW, Smith GR. Conserved and nonconserved proteins for meiotic DNA breakage and repair in yeasts. \u003cem\u003eGenetics\u003c/em\u003e. 2004;167(2):593-605. doi:10.1534/genetics.103.023762\u003c/li\u003e\n\u003cli\u003eLiu Y, Lin Z, Yan J, Zhang X, Tong MH. A \u003cem\u003eRad50\u003c/em\u003e-null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells. \u003cem\u003eDevelopment\u003c/em\u003e. 2024;151(8). doi:10.1242/dev.202312\u003c/li\u003e\n\u003cli\u003eKim S, Yamada S, Li T, et al. Mouse MRE11-RAD50-NBS1 is needed to start and extend meiotic DNA end resection. \u003cem\u003eNat Commun\u003c/em\u003e. 2025;16(1). doi:10.1038/s41467-025-57928-x\u003c/li\u003e\n\u003cli\u003eHayashi M, Chin GM, Villeneuve AM. C. elegans germ cells switch between distinct modes of double-strand break repair during meiotic prophase progression. \u003cem\u003ePLOS Genetics\u003c/em\u003e. 2007;3(11). doi:https://doi.org/10.1371/journal.pgen.0030191\u003c/li\u003e\n\u003cli\u003eGirard C, Roelens B, Zawadzki KA, Villeneuve AM. Interdependent and separable functions of Caenorhabditis elegans MRN-C complex members couple formation and repair of meiotic DSBs. \u003cem\u003eProc Natl Acad Sci USA\u003c/em\u003e. 2018;115(19). doi:10.1073/pnas.1719029115\u003c/li\u003e\n\u003cli\u003eBishop K, Kleckner N. DMCI: A meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. \u003cem\u003eCell\u003c/em\u003e. 1992;69(3):439-456,. doi:10.1016/0092-8674(92)90446-j\u003c/li\u003e\n\u003cli\u003eTran PT, Erdeniz N, Dudley S, Liskay RM. Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae. \u003cem\u003eDNA Repair\u003c/em\u003e. 2002;1(11):895-912. doi:10.1016/s1568-7864(02)00114-3\u003c/li\u003e\n\u003cli\u003eJoshi N, Brown MS, Bishop DK, B\u0026ouml;rner GV. Gradual implementation of the meiotic recombination program via checkpoint pathways controlled by global DSB levels. \u003cem\u003eMolecular Cell\u003c/em\u003e. 2015;57(5):797-811. doi:10.1016/j.molcel.2014.12.027\u003c/li\u003e\n\u003cli\u003eMimitou EP, Yamada S, Keeney S. A global view of meiotic double-strand break end resection. \u003cem\u003eScience\u003c/em\u003e. 2017;335(6320):40-45. doi:10.1126/science.aak9704\u003c/li\u003e\n\u003cli\u003eLi J, Hooker GW, Roeder GS. \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e Mer2, Mei4 and Rec114 form a complex required for meiotic double-strand break formation. \u003cem\u003eGenetics\u003c/em\u003e. 2006;173(4):1969-1981. doi:10.1534/genetics.106.058768\u003c/li\u003e\n\u003cli\u003eHenderson KA, Kee K, Maleki S, Santini PA, Keeney S. Cyclin-Dependent Kinase directly regulates initiation of meiotic recombination. \u003cem\u003eCell\u003c/em\u003e. 2006;125(7):1321-1332. doi:10.1016/j.cell.2006.04.039\u003c/li\u003e\n\u003cli\u003eMaleki S, Neale MJ, Arora C, Henderson KA, Keeney S. Interactions between Mei4, Rec114, and other proteins required for meiotic DNA double-strand break formation in Saccharomyces cerevisiae. \u003cem\u003eChromosoma\u003c/em\u003e. 2007;116(5):471-486. doi:10.1007/s00412-007-0111-y\u003c/li\u003e\n\u003cli\u003eYunus AA, Lima CD. Purification of SUMO Conjugating Enzymes and Kinetic Analysis of Substrate Conjugation. In: Ulrich HD, ed. \u003cem\u003eSUMO Protocols\u003c/em\u003e. Vol 497. Methods in Molecular Biology. Humana Press; 2009:167-186. doi:10.1007/978-1-59745-566-4_11\u003c/li\u003e\n\u003cli\u003eCannavo E, Cejka P, Kowalczykowski SC. Relationship of DNA degradation by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection. \u003cem\u003eProc Natl Acad Sci USA\u003c/em\u003e. 2013;110(18). doi:10.1073/pnas.1305166110\u003c/li\u003e\n\u003cli\u003eMurakami H, Borde V, Nicolas A, Keeney S. Gel electrophoresis assays for analyzing DNA double-strand breaks in Saccharomyces cerevisiae at various spatial resolutions. In: Keeney S, ed. \u003cem\u003eMeiosis\u003c/em\u003e. Vol 557. Methods in Molecular Biology. Humana Press; 2009:117-142. doi:10.1007/978-1-59745-527-5_9\u003c/li\u003e\n\u003cli\u003eDelaglio F, Grzesiek S, Vuister GeertenW, Zhu G, Pfeifer J, Bax A. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. \u003cem\u003eJ Biomol NMR\u003c/em\u003e. 1995;6(3). doi:10.1007/BF00197809\u003c/li\u003e\n\u003cli\u003eVranken WF, Boucher W, Stevens TJ, et al. The CCPN data model for NMR spectroscopy: Development of a software pipeline. \u003cem\u003eProteins\u003c/em\u003e. 2005;59(4):687-696. doi:10.1002/prot.20449\u003c/li\u003e\n\u003cli\u003eSheng W, Liao X. Solution structure of a yeast ubiquitin‐like protein Smt3: The role of structurally less defined sequences in protein\u0026ndash;protein recognitions. \u003cem\u003eProtein Science\u003c/em\u003e. 2002;11(6):1482-1491. doi:10.1110/ps.0201602\u003c/li\u003e\n\u003cli\u003eDosztanyi Z, Csizmok V, Tompa P, Simon I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. \u003cem\u003eBioinformatics\u003c/em\u003e. 2005;21(16):3433-3434. doi:10.1093/bioinformatics/bti541\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7215871/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7215871/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Mre11 nuclease is part of the highly conserved MRX complex involved in the repair of DNA double-strand breaks (DSBs). During meiosis in budding yeast, MRX is also required for the programmed induction of DSBs by Spo11, thereby initiating homologous recombination to promote accurate chromosome segregation. Recruitment of Mre11 to meiotic DSB sites depends on Rec114-Mei4 and Mer2 (RMM), which are thought to organize the meiotic DSB machinery by a mechanism involving biomolecular condensation. Here, we explored the role of Mre11 during meiosis and its relationship to RMM condensation. We show that both Mre11 and MRX complexes form DNA-dependent, hexanediol sensitive condensates \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eIn vivo\u003c/em\u003e, Mre11 assembles into DNA damage-dependent foci in vegetative cells and DSB-independent foci in meiotic cells. \u003cem\u003eIn vitro\u003c/em\u003e condensates and \u003cem\u003ein vivo\u003c/em\u003e foci both depend on the C-terminal intrinsically-disordered region (IDR) of Mre11. Importantly, while the Mre11 IDR is dispensable for vegetative DNA repair it is essential during meiosis. The C-terminal region of Mre11 forms a short α-helix that binds a conserved region of Mer2, and mutating residues within this interface reduces Mre11 foci and DSB formation. Finally, we identified a SUMO-interacting motif within the Mre11 IDR that enhances recruitment of Mre11 during meiosis and facilitates DSB formation. Our results provide new insights into the biophysical properties of Mre11 and its role in initiating meiotic recombination.\u003c/p\u003e","manuscriptTitle":"Recruitment of Mre11 to recombination sites during meiosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 09:50:04","doi":"10.21203/rs.3.rs-7215871/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a052c599-3caf-485a-af06-c51f08701ca9","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52595185,"name":"Biological sciences/Biochemistry/Proteins/DNA-binding proteins"},{"id":52595186,"name":"Biological sciences/Molecular biology/Cell division/Meiosis"},{"id":52595187,"name":"Biological sciences/Molecular biology/DNA damage and repair/Double-strand DNA breaks"}],"tags":[],"updatedAt":"2025-08-19T09:50:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 09:50:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7215871","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7215871","identity":"rs-7215871","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}