DNA-driven condensation assembles the meiotic DNA break machinery

Abstract

The accurate segregation of chromosomes during meiosis-which is critical for genome stability across sexual cycles-relies on homologous recombination initiated by DNA double-strand breaks (DSBs) made by the Spo11 protein^1,2. The formation of DSBs is regulated and tied to the elaboration of large-scale chromosome structures^3-5, but the protein assemblies that execute and control DNA breakage are poorly understood. Here we address this through the molecular characterization of Saccharomyces cerevisiae RMM (Rec114, Mei4 and Mer2) proteins-essential, conserved components of the DSB machinery². Each subcomplex of Rec114-Mei4 (a 2:1 heterotrimer) or Mer2 (a coiled-coil-containing homotetramer) is monodispersed in solution, but they independently condense with DNA into reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive this condensation. Mutations that weaken protein-DNA interactions strongly disrupt both condensate formation and DSBs in vivo, and thus these processes are highly correlated. In vitro, condensates fuse into mixed RMM clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centres of DSB activity. We propose that multilayered control of Spo11 arises from the recruitment of regulatory components and modulation of the biophysical properties of the condensates.

Extended Data Fig. 1:. Characterization of the Rec114–Mei4 complex.

a. Strategy for purification of a hypothetical Rec114–Mei4–Mer2 (RMM) complex. Combinations of MBP-tagged and HisFlag-tagged RMM subunits were co-expressed in insect cells. After cell lysis, complexes were purified by sequential affinity chromatography and analyzed by SDS-PAGE. Expression and solubility of the recombinant proteins are verified by western blotting (WB) of cell extracts. b. Analysis of purified complexes. Rec114–Mei4 complexes were apparent (lanes 1 and 3), but no Mer2 was co-purified. Lanes 2 and 4 show some enrichment of MBP-Mer2, but no co-purification of Rec114–Mei4. The presence of MBP-Mer2 in lanes 2 and 4 of the silver-stained gel may be due to background binding of MBP-Mer2 to the NiNTA resin (potentially via adsorption of DNA to the resin), or to low-affinity interactions to immobilized His-tagged Rec114–Mei4 complexes. Either way, none of the combinations tested yielded stoichiometric complexes of all three RMM subunits. Western blot controls of cell extracts showed that the tagged RMM proteins were expressed and soluble. c. Mass spectrometry analysis of Rec114–Mei4 complexes. Purified Rec114-Mei4 complexes were treated with trypsin and analyzed by LC-MS/MS. The ratio of spectral counts between Rec114 and Mei4 provides additional evidence supporting the 2:1 stoichiometry of the complex. d, e. Alignments and predicted secondary structures of the C-terminus of Rec114 (d) and the N-terminus of Mei4 (e). The positions of the conserved SSMs are indicated. f. Cartoon of the Rec114–Mei4 truncations analyzed. g. Purification of Rec114–Mei4 truncations. Proteins were expressed in E. coli and purified on NiNTA resin using a HisSUMO tag fused to the N-terminus of the Rec114 fragment. After removal of the tag by treatment with the SUMO protease Ulp1, complexes were further purified by gel filtration. A Coomassie-stained SDS-PAGE analysis of purified complexes is shown. 5 μg was loaded for each sample. Polypeptides containing Rec114^(375−428) and Mei4⁽¹⁻⁴³⁾ retained the ability to interact (combination #4). h. SEC-MALS analysis of Rec114–Mei4 truncations. The data are consistent with expectation for truncations that contain two Rec114 subunits and one Mei4 subunit. The C-terminus of Rec114 alone forms a dimer. i. Wild type and F411A-containing variants of ^HisSUMORec114^(325−428) were co-expressed with Mei4⁽¹⁻⁹⁰⁾ and purified by chromatography on NiNTA resin. The absence of the Mei4 fragment with Rec114-F411A shows that the mutation abolishes the interaction with Mei4. j. SEC-MALS analysis of untagged wild-type (WT, reproduced from panel H to aid comparison) and F411A Rec114^(325−428) show that the mutation affects Rec114 dimerization. k. Y2H analysis of the interaction of Gal4BD-Rec114 (WT and F411A) with LexA-Mei4, LexA-Rec102, or LexA-Rec104 (mean and SD from four replicates). β-Gal units are quantified based on hydrolysis of ONPG. The F411A mutation abolishes the interaction of Rec114 with Mei4, but not with Rec102 and Rec104. l. Southern blot analysis of meiotic DSB formation at the CCT6 hotspot, showing that rec114-F411A is defective in meiotic DSB formation. m. Spore viability of rec114-F411A mutant (n = 40). n. Western-blot analyses of meiotic protein extracts from myc-tagged REC114-WT and F411A strains. The F411A mutation does not compromise the expression of Rec114. o. Immunofluorescence microscopy analysis of meiotic chromosome spreads with wild-type and F411A myc-tagged Rec114. Green, anti-myc; red, synaptonemal complex component Zip1; blue, DNA. Quantification of the number of Rec114 foci per leptotene or early zygotene cell is plotted; error bars show mean ± SD (n = 20 and 38 cells for WT and F411A, respectively). The F411A mutation abolishes the formation of chromatin-associated Rec114 foci.

Extended Data Fig. 2:. DNA-binding properties of Rec114–Mei4 and Mer2 complexes.

a, b. Gel shift analysis of Rec114–Mei4 (a) or Mer2 (b) binding to 20- or 40- bp DNA substrates. Quantification is in Fig. 2b. c, d. Competition assay of Rec114–Mei4 (c) or Mer2 (d) binding to 80 bp radiolabeled DNA (1 nM) in the presence of 20- or 80 bp cold competitor. Fold excess is in nucleotides. Lines are one-phase decay fits. e, f. Binding to plasmid DNA analyzed by native agarose gel electrophoresis. Rec114–Mei4 (e) and Mer2 (f) were titrated with 2 nM plasmid DNA (pUC19) in the presence or absence of 5 mM MgCl₂. Rec114–Mei4 complexes bound with roughly similar affinity independently of the presence of Mg²⁺ (apparent K_D ≈ 50–80 nM). Note that the apparent affinity is significantly lower than suggested by the gel shift analyses with radiolabeled substrates presented in panel a and Fig. 2a, b (see apparent affinities in Fig. 2 legend). We interpret that this difference is because the proteins coalesce on a small fraction of the plasmid molecules, as illustrated in the cartoon below. Indeed, bound plasmids remained trapped in the wells, which is consistent with cooperative assembly of large nucleoprotein structures. Because each plasmid substrate provides many more binding sites than the short oligonucleotide substrates in panel a and Fig. 2a, a higher concentration of protein is required to reach complete binding of all of the plasmid molecules. In contrast to Rec114–Mei4, Mer2 showed efficient binding in the absence of Mg²⁺ in this assay (K_D = 30 ± 2 nM) but binding appeared to be considerably inhibited in the presence of Mg²⁺ (K_D ≈ 150 nM), as indicated by the persistence of unbound substrate at high protein concentrations. However, while the electrophoretic mobility of Mer2-bound plasmids decreased steadily as the concentration of Mer2 increased in the absence of Mg²⁺, no such steady progression was observed when Mg²⁺ was included. Instead, a minority of bound substrates shifted to a low-mobility species (labeled * in panel f, bottom), indicating that they were occupied by multiple Mer2 complexes. We interpret that, rather than inhibiting DNA binding, Mg²⁺ promotes cooperativity, in agreement with the fluorescence microscopy analysis (Extended Data Fig. 3b). The difference in migration distance of the plasmid between the +/− Mg²⁺ gels is due to the presence of Mg²⁺ in the electrophoresis buffer. g. AFM imaging of 12 nM Rec114–Mei4 in the absence (left) or in the presence (right) of 1 nM plasmid DNA (pUC19).

Extended Data Fig. 3:. Properties of Rec114–Mei4 and Mer2 DNA-dependent condensates.

a, b. Visualization of nucleoprotein condensates by epifluorescence microscopy using tagged Rec114–Mei4 (a) or Mer2 (b) in the presence or absence of 5 mM MgCl₂. Foci were defined using a fixed intensity threshold between samples. Each point represents the measurement from a field of view. Error bars show mean ± SD from (a) 10 fields of view of 1.7 × 10⁴ μm² or (b) 27 and 26 sections of 400 μm² with and without Mg²⁺, respectively. c-f. Effect of fluorophore labeling or tagging on the DNA-binding and DNA-driven condensation activities of Rec114–Mei4 and Mer2 complexes. Labeling with Alexa594 or Alexa488 was achieved using amine-reactive fluorophores. Tagging was achieved by fusion of Rec114 with the monomeric fluorescent protein mScarlet or fusion of Mer2 with the weakly dimerizing fluorescent protein eGFP. The results described here indicate that the covalent Alexa labeling has little if any effect on DNA binding properties of these complexes, whereas fluorescent protein tagging caused subtle alterations in DNA binding and/or condensation. In most subsequent experiments, we used the dye-labeled complexes to minimize steric effects or oligomerization of fluorescent protein tags. c. Gel-shift analysis of binding of unlabeled, Alexa594-labeled, or mScarlet-tagged Rec114–Mei4 complexes to an 80-bp radiolabeled DNA substrate. The three versions of the Rec114–Mei4 complex have the same intrinsic DNA-binding activity. d. Gel-shift analysis of binding of unlabeled, Alexa488-labeled, or eGFP-tagged Mer2 complexes to an 80-bp radiolabeled DNA substrate. The DNA-binding activity of the Alexa-labeled Mer2 complex is nearly identical to the untagged protein, but the eGFP-tagged complex has 3.5-fold reduced DNA-binding activity. e. A comparison between Alexa-labeled and mScarlet-tagged Rec114–Mei4 complexes for DNA-driven condensation. Focus numbers (left graphs) and total fluorescence intensity within foci normalized to the no-PEG samples (right graphs) are shown for the complexes in the presence or absence of 5% PEG. With and without PEG, mScarlet-tagged Rec114–Mei4 produced more foci than the Alexa-labeled version. Because intrinsic DNA binding was indistinguishable between the complexes (panel c), we infer that the mScarlet-tagged complexes had a reduced efficiency in the cooperative formation of large condensates compared to the Alexa-labeled version, producing more numerous foci. Asterisk indicates p < 0.0001 (two-tailed t test). Lines and error bars are mean ± SD from 8–10 fields of view. f. A comparison between Alexa-labeled and eGFP-tagged Mer2 complexes for DNA-driven condensation. Quantification is presented as in panel e. The two labeled complexes show different numbers and intensities of foci in the presence of PEG. It is likely that the DNA-binding defect of the eGFP construct (panel d) leads to the formation of fewer, brighter condensates. It is possible that the weak dimerization activity of eGFP also contributes. Asterisk indicates p < 0.0001 (two-tailed t test). Lines and error bars are mean ± SD from 9–10 fields of view. g, h. Effect of a crowding agent (PEG) on formation of nucleoprotein condensates visualized using covalently fluorophore-labeled Rec114–Mei4 (g) or Mer2 (h). Graphs show the effect of protein concentration on DNA-driven condensation in the presence or absence of 5% PEG. Left graphs show focus numbers and right graphs show the total fluorescence intensity within foci (normalized to the mean of the highest intensity sample). Points and error bars are means ± SD from 4–6 fields of view (g) or 7–10 fields of view (h). The titrations reveal complex behaviors: (g) In the presence of PEG, titration of Rec114–Mei4 from 4 to 32 nM led to a steady decrease in the number of foci, which was accompanied by a concomitant increase in focus intensity. In the absence of PEG, however, the number of Rec114–Mei4 foci first peaked at 8 nM before decreasing as the intensity of the foci started to increase. Nevertheless, focus intensity plateaued at a much lower intensity than in the presence of PEG. (h) In the case of Mer2, titration from 25 to 300 nM in the presence of PEG yielded a peak in the number of foci at ~100 nM, which then sharply declined and stabilized beyond 150 nM. Consistently, Mer2 foci remained at a constant, low intensity between 25 and 100 nM, then became abruptly brighter above 100 nM. In the absence of PEG, the number of Mer2 foci increased between 25 and 200 nM, then started to decrease beyond that threshold. These behaviors likely reflect complex combined effects of nucleation, growth, and collapse of the condensates, which are each affected differently by protein concentrations and by the crowding effect provided by PEG. See Source Data for exact n values for panels e, f, g, and h.

Extended Data Fig. 4:. Properties of Rec114–Mei4 and Mer2 DNA-dependent condensates.

a, b. Effect of challenging Rec114–Mei4 (a) or Mer2 (b) nucleoprotein condensates with DNase I or 0.5 M NaCl. Condensates were assembled for 5 minutes prior to challenge. Quantification is provided of focus numbers per 1000 μm² and of the total fluorescence intensity within foci within fields of view (normalized to mean of the no-treatment controls). Error bars show mean ± SD from 5–10 fields of view. c, e. Titrations of Rec114–Mei4 (c) and Mer2 (e) in the presence of DNA and PEG and various concentrations of NaCl. Heat maps represent the fraction of fluorescence signal found within foci. Condensed fractions are maximal at high protein and low salt concentrations. At all protein concentrations, condensation is essentially abolished beyond 250 mM NaCl. This suggests that electrostatic interactions, likely between the negatively charged DNA backbone and positively charged protein residues, are important for condensation. d, f. Time dependence for irreversibility of Rec114–Mei4 (d) and Mer2 (f) condensates. Some phase-separated liquid droplets have been shown to mature over time and progressively adopt gel-like or solid states^,–. Such sol-gel transitions may occur spontaneously through different mechanisms, including fibrillization and entanglement, and are thought to be counteracted in vivo to prevent the progressive accumulation of amyloid-like structures associated with pathological states. To address whether our condensates are prone to progressive hardening, we queried the effect of assembly time on reversibility. We performed a time-course experiment where the condensates were challenged by treatment with 0.5 M NaCl after an indicated period of assembly in the presence or absence of PEG. The graph shows the total intensity summed for foci within fields of view, expressed as a percentage of the intensity without a salt challenge. Points and error bars are means ± SD for 6–10 fields of view. With Rec114–Mei4, 10% and 50% of fluorescent signal became refractory to the salt wash within 5 minutes of incubation time in the absence and presence of PEG, respectively (see panel a for example images and quantification). With Mer2, there was no evidence for the formation of irreversible structures in the absence of PEG during the course of the experiment. However, up to 25% of the focus intensity resisted the salt wash treatment after 8 minutes of incubation time in the presence of PEG. Therefore, both Rec114–Mei4 and Mer2 have a propensity to form more stable, perhaps gel-like, structures over time. Under our experimental conditions, this was more evident for Rec114–Mei4 than for Mer2, and was accentuated by molecular crowding. g, h. Assembly of Rec114–Mei4 (g) and Mer2 (h) with fluorescently labeled 9.6 kb and 100 bp linear DNA substrates. The overlap between the protein foci and puncta of DNA shows that the DNA is also enriched in the condensates. However, in contrast to the protein signal, the fluorescent signal of the DNA covers the slide because DNA is in excess and does not condense by itself. i, j. Competition between long and short DNA substrates for incorporation into condensates. Rec114–Mei4 (i) or Mer2 (j) condensates were assembled in the presence of a fluorescently labeled DNA substrate with or without 20-fold nucleotide excess of unlabeled competitor. The amount of fluorescent DNA signal averaged between ten foci is plotted. In each case, the 9.6-kb substrate was a more effective competitor than the 100-bp substrate. In addition, the 100-bp substrate was more successful at competing with the 100-bp fluorescent substrate than with the 9.6-kb fluorescent substrate. This preference for large DNA substrates is consistent with the hypothesis that the condensates form through multivalent interactions between the positively charged residues of Rec114-Mei4 or Mer2 and the sugar-phosphate backbone of the DNA. See Source Data for exact n values for panels a, b, d, and f.

Extended Data Fig 5:. Growth of DNA-driven condensates by fusion.

a. Three scenarios for the assembly of DNA-driven condensates. (i) Nucleation could be limiting, with focus growth resulting principally from incorporation of protein from soluble pools. (ii) Frequent nucleation events could occur initially leading to large numbers of small foci, whereupon some foci dissolve and others grow. (iii) Frequent nucleation could yield numerous small foci that then collide and fuse to yield fewer, larger foci. See Supplementary Discussion 1 for more detail. b. Time course of the assembly of Rec114–Mei4 foci in the presence of plasmid DNA. The x axis indicates the time in solution before plating, upon which DNA is immobilized to the glass slide while soluble protein is still free to diffuse. Quantification is provided of focus numbers and average focus intensity (normalized to the mean at 30 min). Error bars show mean ± SD from 10 fields of view. c. FRAP experiments with Mer2 and Rec114–Mei4 condensates. Points and error bars are mean ± SD for six photobleached condensates.

Extended Data Fig 6:. Identification of DNA-binding residues and effect of DNA binding on condensation in vitro and in vivo and on Spo11-induced break formation.

a. Mapping the DNA-binding domain of Rec114–Mei4 complexes. Gel-shift analysis was performed with pUC19 plasmid DNA and the Rec114–Mei4 protein constructs shown in Extended Data Fig. 1f. Constructs #2, #3 and #4, which include the C terminus of Rec114 and the N terminus of Mei4, were competent for DNA binding. The difference in mobility of shifted species between these constructs is in line with the difference in sizes of the protein complexes. Mei4 is dispensable for DNA binding by Rec114 (Construct #5 lacks Mei4). The N terminus of Rec114 alone, encompassing the PH domain, did not bind DNA (Construct #6). None of the constructs showed evidence for cooperative DNA binding (unlike the full-length protein, see Extended Data Fig. 2e), suggesting that they do not undergo DNA-driven condensation. b. Gel shift analysis wild-type (WT) and mutant Rec114–Mei4 complexes binding to an 80-bp DNA substrate. The Rec114–4KR mutant has residues R395, K396, K399, and R400 mutated to alanine. Lines on graphs are sigmoidal curve fits. c. Mapping the DNA-binding domain of Mer2. Gel-shift analysis was performed with pUC19 plasmid DNA and HisSUMO-tagged Mer2 protein that was either full-length (FL), had the N terminus removed (fragment 77–314), or had both the N and C termini removed (fragment 77–227). Deleting the N terminus alone had no significant effect on DNA binding, but further deleting the C terminus strongly reduced DNA binding. d. Effect of the Rec114–4KR mutation on condensation in vitro. Reactions included 5% PEG. Each point is the average of the intensities of foci in a field of view (n = 20 fields), normalized to the overall mean for wild type. Error bars show mean ± SD. e. Incorporation of Mer2-KRRR into preformed condensates. Condensates were assembled with 100 nM unlabeled Mer2. Reactions were then supplemented with the indicated amount eGFP-Mer2 (WT or KRRR) and plated immediately. Incorporation of eGFP-tagged complexes within condensates was quantified. Points and error bars are mean ± SD from 20 fields of view. f. Immunofluorescence on meiotic chromosome spreads for myc-tagged Rec114. The number of foci per leptotene or early zygotene cell is plotted. Error bars show mean ± SD (n = 44 and 40 cells for WT and 4KR, respectively). g. Immunoblotting of meiotic protein extracts for wild type and mutant Rec114 (left) or Mer2 (right). h. Partial proteolysis of wild-type and mutant Mer2 and Rec114–Mei4 complexes. i. Immunoblot analysis of Mer2-WT and Mer2-KRRR. Protein extracts of meiotic time courses were analyzed by SDS-PAGE followed by immunoblotting against Mer2-myc. Anti-Kar2 was used as a loading control. Quantification of immunoblot signal is plotted. Mer2^myc-KRRR reached higher steady-state protein levels and persisted longer than wild-type Mer2^myc. A previous study showed that mutating an essential CDK phosphorylation site of Mer2 (Ser30) or inhibiting CDK activity led to reduced turnover of Mer2, similar to the effect of the KRRR mutant. This is consistent with the hypothesis that Mer2 turnover is tied to phosphorylation, which requires DNA binding. j. Southern blot analysis of meiotic DSB formation at the CCT6 hotspot in strains expressing wild-type or mutant Rec114 protein. k. Labeling of Spo11-oligo complexes in wild type and mutant Rec114 (top) and Mer2 (bottom) strains. Error bars represent the range from two biological replicates. l. Spore viability of wild type and mutant Rec114 (left) and Mer2 (right) strains (n = 40). For gel source data, see Supplementary Figure 1.

Extended Data Fig. 7:. Rec114–Mei4 and Mer2 form mixed condensates.

a. Rec114–Mei4 colocalizes with Mer2 in mixed condensates irrespective of DNA concentration. Reactions containing 16 nM Rec114–Mei4 and 100 nM Mer2 in the presence of 1, 10, or 100 ng/μl plasmid DNA were assembled for 20 minutes at 30 °C. DAPI (5 μg/ml) was added to the reaction before applying to glass slides. DNA enrichment within the condensates is visible at lower DNA concentrations (top and middle rows), but is not as clear at high DNA concentrations (bottom row). The ratios of Rec114–Mei4 (heterotrimers) and Mer2 (tetramers) to each 2.6-kb plasmid DNA molecule are indicated on the right. Colocalization of Rec114–Mei4 and Mer2 complexes is evident even with a molar excess of DNA molecules, demonstrating that formation of joint foci is not simply because both protein complexes are independently associating with a limiting number of DNA substrates. b. Correlated intensity of Rec114–Mei4 and Mer2 proteins within the condensates. Each point shows the fluorescence intensity in an individual focus (n = 950, 925 and 1000 foci from 2–3 fields of view for samples with 1, 10 and 100 ng/μl DNA, respectively), normalized to the average foci intensity per field of view. The strong correlation indicates that the composition of the condensates is highly uniform between foci. In the presence of high DNA concentration, the fraction of smaller foci increased and correlated intensities decreased. c. Recruitment of soluble Rec114–Mei4 (left) or Mer2 (right) into preassembled condensates of Mer2 (left) or Rec114–Mei4 (right). White arrowheads point to examples of the preassembled condensates. d. Pulldown of purified Mer2 on amylose resin with or without immobilized Rec114–Mei4 complexes. e. XL-MS of Rec114–Mei4–Mer2 condensates (620 crosslinked peptides, 229 distinct crosslinked pairs of lysines).

Extended Data Fig. 8:. Recruitment of the Spo11 core complex to Rec114–Mei4–Mer2 condensates.

a. Quantification of core complex signal within Rec114–Mei4 foci in the presence (100 nM) or absence of Mer2. The average intensity within 20 foci is plotted for each reaction. Shaded areas represent 95% confidence intervals. b. Quantification of core complex signal within Mer2 foci in the presence (16 nM) or absence of Rec114–Mei4. Reactions contained 25 nM Mer2. The average intensity within 20 foci is plotted for each reaction. Shaded areas represent 95% confidence intervals. c. Effect of including 100 nM ^MBPRec102–Rec104^HisFlag competitor on the recruitment of the core complex to RMM condensates (16 nM Rec114–Mei4, 100 nM Mer2). The fraction of Rec114–Mei4–Mer2 foci that contain detectable core complex signal is plotted (mean and SD from 10 fields of view). d. Intensity of core complex signal within Rec114–Mei4–Mer2 condensates in the absence or presence of Rec102–Rec104 competitor. The average core complex intensity within 20 foci is plotted for each reaction. Shaded areas represent 95% confidence intervals. e. Mapping regions of Rec114 required for interaction with Rec102 or Rec104 by Y2H analysis. β-galactosidase units are measured for the interaction between truncated variants of Gal4AD-Rec114 and LexA-Rec102 or LexA-Rec104 (mean and SD from four replicates). The position of the HLS mutation within the Rec114 PH-fold is indicated. f. Impact of the HLS mutation on the formation of comingled RMM condensates. Error bars show mean ± SD from 10 fields of view. g. Spore viability of Rec114-WT and HLS mutant strains. h. Immunoblot analysis of meiotic protein extracts from myc-tagged Rec114-WT and HLS mutant strains. Samples from two biological replicates are shown. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 9:. A condensate model for assembly of the meiotic DSB machinery and implications for the control of DSB formation and repair.

a. Assembly of the DSB machinery. (Left) Rec114–Mei4 and Mer2 complexes bind DNA in a highly cooperative manner to form large mixed nucleoprotein condensates. (Right) These condensates provide a platform to recruit the core complex through interactions that involve the N-terminal domain of Rec114 and the Rec102–Rec104 components of the core complex. Multiple Spo11 complexes are recruited and may engage an incoming DNA loop simultaneously. The molecular arrangement of the core complex proteins is based on ref . See Supplementary Discussion 4 for more detail. b. Hotspot competition and DSB interference. Competition arises prior to DSB formation as a consequence of the partitioning of RMM proteins into condensates. DSB interference is implemented through local inhibition of further DSB formation by DSB-activated Tel1. Inhibition could work on the same cluster that generated the activation DSB as well as on nearby clusters in cis. See Supplementary Discussion 5 for more detail. c. The coherence provided by the condensates may serve functions during repair, including the maintenance of a physical connection between the DNA ends that involves end-capping by condensate-embedded core complexes. See Supplementary Discussion 6 for more detail.

Fig. 1:. Purification and subunit arrangement of the S. cerevisiae RMM proteins.

a. Prediction of protein disorder (IUPRED server). The ANCHOR score predicts the transition from unstructured to structured depending on a binding partner. Previously identified SSMs are highlighted^,. b. SDS-PAGE of purified tagged and untagged Rec114–Mei4 complexes. 4 μg was loaded. c. SEC-MALS analysis of tagged and untagged Rec114–Mei4. The traces show UV absorbance (left axis), circles are molar mass measurements across the peak (right axis). Elution positions of protein standards are marked. d. XL-MS analysis of Rec114–Mei4 (4812 crosslinked peptides, 258 distinct crosslinked pairs of lysines). Black loops are intermolecular self-links. Black vertical lines indicate lysines. e. Cartoon of the Rec114–Mei4 complex. f. Protein disorder prediction for Mer2. The predicted coiled coil and previously identified SSMs are highlighted^,. g. SDS-PAGE of purified Mer2. 4 μg was loaded. h. SEC-MALS analysis of Mer2. i. XL-MS analysis of Mer2 (487 crosslinked peptides, 89 distinct crosslinked pairs of lysines). j. SEC-MALS analysis of the coiled coil domain of Mer2 and a single-chain dimer variant of the coiled coil domain. A tetramer of monomers and a dimer of single-chain dimers both have an expected MW of 70 kDa. The difference between the profiles of the monomer and single chain dimer can be explained by reduced degrees of freedom (tension) in the single-chain dimer and heterogeneity. k. Interpretive cartoon of the molecular arrangement of the coiled coil domain of Mer2. For gel source data, see Supplementary Figure 1.

Fig. 2:. Rec114–Mei4 and Mer2 form condensates on DNA.

a. Gel shift analysis of Rec114–Mei4 and Mer2 binding to 80 bp DNA substrates (see also Extended Data Fig. 2a, b). b. Quantification of gel-shift analyses with 20-, 40- or 80 bp substrates. Error bars are ranges from two independent experiments. Lines are sigmoidal curves fit to the data, except for the 20 bp substrate (smooth spline fits). Apparent affinities of Rec114–Mei4 are: 6 ± 1.4 nM (80 bp, mean and range); 35 ± 1.3 nM (40 bp); ≈ 80 nM (20 bp). Apparent affinities of Mer2 are: 19 ± 1.5 nM (80 bp); 64 ± 15 nM (40 bp); > 400 nM (20 bp). Here and elsewhere, concentrations for Rec114–Mei4 refer to the trimeric complex, but for Mer2 they refer to the monomer. Therefore, the complexes have comparable affinities for DNA if the quaternary units (trimers and tetramers, respectively) are considered. c. AFM imaging of 50 nM Mer2 in the absence (left) or presence (right) of 1 nM plasmid DNA (pUC19). d. Time course of the assembly of Mer2 foci in the presence of plasmid DNA. The x axis indicates the time in solution before plating, upon which DNA is immobilized to the glass slide while soluble protein is still free to diffuse. Quantification is provided of focus numbers and average focus intensity (normalized to the mean at 30 min). Error bars show mean ± SD from 8–10 fields of view (see Source Data for exact n values). For gel source data, see Supplementary Figure 1.

Fig. 3:. DNA binding by Mer2 is important for macromolecular condensation in vitro and in vivo and for Spo11-induced break formation.

a. Gel shift assay of wild-type (WT) and mutant Mer2 complexes binding to an 80-bp DNA substrate. The Mer2-KRRR mutant has residues K265, R266, R267, and R268 mutated to alanine. Lines on the graph are a sigmoidal curve (WT) and a smooth spline (KRRR) fit to the data. b. Effect of the Mer2-KRRR mutation on condensation in vitro. Reactions included 5% PEG. Each point is the average of the intensities of foci in a field of view, normalized to the overall mean for wild type. Error bars show mean ± SD (n = 20 fields of view). c. Immunofluorescence on meiotic chromosome spreads for myc-tagged Mer2. The number of foci per leptotene or early zygotene cell is plotted. Error bars show mean ± SD (n = 48 and 95 cells for WT and KRRR, respectively). d. Southern blot analysis of meiotic DSB formation at the CCT6 hotspot in wild type and mer2 mutant strains. For gel source data, see Supplementary Figure 1.

Fig. 4:. Tripartite Rec114–Mei4–Mer2 nucleoprotein condensates recruit the Spo11 core complex.

a. Fluorescently labeled Rec114–Mei4 (17 nM) and Mer2 (100 nM) were mixed prior to DNA-driven condensation (for 30 minutes, 5.6 nM pUC19) and imaging by epifluorescence microscopy. b. Rec114–Mei4 and Mer2 nucleoprotein condensates were assembled separately for 10 minutes then mixed. After mixing, reactions contained 5.6 nM pUC19, 8.5 nM ^Alexa594Rec114–Mei4 and 50 nM ^Alexa488Mer2. Samples were dropped on a microscope slide 10 seconds (top) or 20 minutes (bottom) after mixing. White arrowheads indicate Mer2 condensates. c. Time course of Rec114–Mei4 and Mer2 colocalization. The time to achieve 50% of Mer2 foci overlapping with Rec114–Mei4 is indicated (t_1/2). Lines are one-phase association models fit to the data. Error bars show mean ± SD from 9–10 fields of view. d. Incorporation of Alexa488-labeled core complexes into Alexa594-labeled Rec114–Mei4–Mer2 condensates. e. Fraction of Rec114–Mei4 foci that contained detectable core complex signal as a function of Mer2 concentration. Error bars show mean ± SD from 10 fields of view. f. Fraction of Mer2 foci that contained detectable core complex signal as a function of Rec114–Mei4 concentration. Error bars show mean ± SD from 9–10 fields of view. g. Y2H interaction between Gal4AD-Rec114 wild type or H39A/L40A/S41A (HLS) mutant and LexA-Mei4, LexA-Rec114, LexA-Rec102, or LexA-Rec104 (mean and SD from four replicates). h. Southern blot analysis of meiotic DSB formation at the CCT6 hotspot. i. Immunofluorescence microscopy of meiotic chromosome spreads with myc-tagged Rec114-WT and HLS mutant strains. Green, anti-myc (Rec114); red, anti-Zip1; blue, DAPI. Quantification of the number of Rec114 foci per leptotene or early zygotene cell is plotted (n = 24). Line and error bars represent mean ± SD. For gel source data, see Supplementary Figure 1. See Source Data for exact n values for panels c and f.