Extended Data Fig. 3: Recombination intermediates containing dHJs and SEIs are stable during pachytene.

a, Representative in situ immunofluorescence images of cells at indicated times in SPM after Rec104^FRB anchor-away by rapamycin addition (or DMSO as control) at 7 h, immunostained for the HA epitope to visualize the subcellular localization of Rec104^FRB. DNA is visualized with DAPI (grey). b, Quantification of cells with nuclear Rec104^FRB at indicated times in SPM from (a) (n = 100 cells per time point). c, Southern blot analysis of recombination at the HIS4::LEU2 recombination hotspot from the experiment described in Fig. 1h. Two biological replicates are shown; replicate 1 corresponds to the cropped blot in Fig. 1i. P1, parental 1; P2, parental 2; CO1, crossover 1; CO2, crossover 2; DSBs, double-strand breaks; JMs, DNA joint molecules. Asterisk indicates meiosis-specific recombinant band from gene conversion of the XhoI site closest to the DSB site. Dagger indicates ectopic recombination bands between HIS4::LEU2 and leu2::hisG at the native LEU2 locus. d, Native/native two-dimensional Southern blot analysis of branched JMs at the HIS4::LEU2 recombination hotspot from indicated time points of replicate 1 in (c). The different DNA JM species are depicted in the left blot image. SEI, single-end intermediate; IH-dHJ, interhomologue-dHJ; IS-dHJ, intersister-dHJ; mcJM, multichromatid DNA JM. e, Quantification of SEI, IH-dHJ, IS-dHJ and mcJM levels from (d), shown as the percentage of total DNA. f, Western blot analysis of Yen1^ON protein levels in cells from indicated times in SPM of replicate 1 in (c). Crm1 served as protein loading control. Representative of two biological replicates. g, Quantification of Yen1^ON protein levels from the Western blot in (f) and a biological replicate, normalized to the highest value (red line), along with mean JM levels from both replicates in (c) (grey line). Error bars represent the range.