Rad51 and Dmc1 form structures inconsistent with asymmetric loading at individual meiotic DSBs.

(A-E) Rad51 focus pairing is distinct from the spatial arrangement of Zip1. (A-C) Micrographs of wild type diploid leptotene (3 hr) nuclei included in the analysis in (E). Rad51 staining is shown in green, Zip1 staining in red, and DAPI in blue. (D) Micrograph of nucleus excluded from analysis in (E) because of non-punctate Zip1 structures (arrowheads). (E) Nearest neighbor distributions for Zip1 (blue), simulated Zip1 (red), and Rad51 (grey). Sample size is 86 nuclei containing 2,953 Rad51 foci and 1,041 Zip1 foci. Scale bar in panel A is 1 μm. (F-L) Characterization of Rad51-Dmc1 structures in spo11 diploids heterozygous for the VDE cut site. Rad51 staining is shown in green, Dmc1 in red, and DAPI in blue. (F-H) Micrographs of wild type nuclei with (F) a single focal structure (category I), (G) paired foci (category II), and (H) a more complicated structure (>2 Rad51 and/or Dmc1 foci; category III; categories shown in parentheses at top left of each micrograph). (I) Micrograph of mnd1 strand exchange mutant with more complicated structures containing >2 Rad51 and/or Dmc1 foci in close proximity (category III). (J) Micrograph of spatially distinct structures each composed of a single focus (category IV) in an mnd1 mutant. (K) Micrograph of a nucleus with >2 Rad51 and/or Dmc1 structures that are also spatially separated (category V) in an mnd1 mutant. Scale bar = 1 μm, 400 nm for inset. (L) Distribution of these structures in wild type and mnd1 nuclei represented as a percentage of focus-positive nuclei. Micrographs from cultures 4.5 hours after meiotic induction. 116 wild type and 89 mnd1 focus-positive nuclei are included. (M-O) Paired Dmc1 foci can be observed at the HIS4::LEU2 DSB hotspot. Micrographs of paired Dmc1 foci near the lacO (ARS 308) and tetO (ARS 304) landmarks flanking the HIS4::LEU2 DSB hotspot. Dmc1 is shown in Red, tetO in magenta, and lacO in cyan. Scale bar = 500 nm. (O) Micrograph in which sister chromatids are split as indicated by a pair of tetO foci. Micrographs from cultures 2.5 hours after meiotic induction.