Extended Data Fig. 4: Properties of Rec114–Mei4 and Mer2 DNA-dependent condensates.
From: DNA-driven condensation assembles the meiotic DNA break machinery
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 min before challenge. Bottom, quantification of focus numbers per 1,000 μm2 and of the total fluorescence intensity within foci within fields of view (normalized to mean of the no-treatment controls). Mean ± s.d. 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, probably 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 states35,37,38,39. 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 states35. 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 in which 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 graphs show the total intensity summed for foci within fields of view, expressed as a percentage of the intensity without a salt challenge. Mean ± s.d. for 6–10 fields of view. With Rec114–Mei4, 10% and 50% of fluorescent signal became refractory to the salt wash within 5 min of incubation time in the absence and presence of PEG, respectively (see 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 min 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 labelled 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 labelled DNA substrate with or without 20-fold nucleotide excess of unlabelled 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 a, b, d, f.
