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 (Extended Data Fig. 2e)), suggesting that they do not undergo DNA-driven condensation. b, Gel shift analysis of wild-type 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 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. Mean ± s.d. e, Incorporation of Mer2(KRRR) into preformed condensates. Condensates were assembled with 100 nM unlabelled Mer2. Reactions were then supplemented with the indicated amount eGFP–Mer2 (wild-type or KRRR) and plated immediately. Incorporation of eGFP-tagged complexes within condensates was quantified. Mean ± s.d. 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. Mean ± s.d. (n = 44 and 40 cells for wild-type and rec114^4KR strains, 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 wild-type Mer2 and Mer2(KRRR). Protein extracts of meiotic time courses were analysed by SDS–PAGE followed by immunoblotting against Mer2–myc. Anti-Kar2 was used as a loading control. Quantification of immunoblot signal is plotted. Mer2(KRRR)–myc 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 mutant¹⁵. 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, Labelling of Spo11–oligo complexes in wild-type and mutant Rec114 (left) and Mer2 (right) strains. Points represent two biological replicates. l, Spore viability of strains expressing wild-type or mutant Rec114 (left) and Mer2 (right) (n = 40). For gel source data, see Supplementary Fig. 1.

Source data