Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break

Abstract

The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs). Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments.

Fig 1. Rad51-Dmc1 co-foci occur in pairs separated by 200–400 nm.

(A) Spread wild type diploid [WT (2N)] nucleus stained for Rad51 (green) and Dmc1 (red). (B,C,D) Spread spo11 hypomorphic tetraploid [spo11 hypo (4N)] nuclei with relatively low densities of Rad51-Dmc1 staining. Arrowheads indicate examples of paired co-foci in less densely stained areas, magnified in the insets. Scale bar = 1 μm, 400 nm for the inset. (E) Schematic of distance to nearest neighbor measurement for Rad51-to-Rad51 (left) and Rad51-to-Dmc1 (right). Solid arrow represents the distance to the reference focus’ nearest neighbor. Dashed arrows represent the longer distances to other foci. (F,G,H,I) Distribution of nearest neighbor measurements in wild type spo11 hypomorphic tetraploid. Experimental data (red) and matched random simulations (blue) shown for (F) Rad51→Rad51, (G) Dmc1→Dmc1, (H) Rad51→Dmc1, and (I) Dmc1→Rad51. Micrographs from cultures 2.5 hours after meiotic induction. Scoring of 195 focus-positive nuclei, of all staining densities, containing a total of 13,528 Rad51 foci and 13,230 Dmc1 foci are included in the histograms.

Fig 2. 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.

Fig 3. The paired architecture of Rad51-Dmc1 co-foci is independent of strand exchange and synapsis.

(A,B) Micrographs of (A) mnd1 and (B) zip1 mutant nuclei in the spo11 hypomorphic tetraploid background. Scale bar = 1 μm, 400 nm for the inset. Rad51 staining is shown in green, Dmc1 in red, and DAPI in blue. (C-F) Accompanying distribution of nearest neighbor measurements as in Fig 1 for (C) Rad51→Rad51, (D) Dmc1→Dmc1, (E) Rad51→Dmc1, and (F) Dmc1→Rad51. Micrographs from cultures 2.5 hours after meiotic induction. Wild type data repeated from Fig 1 for comparison. Scoring of 70 and 44 focus-positive nuclei; 6,364 and 2,824 Rad51 foci; and 6,114 and 2,655 Dmc1 foci are included in histograms for mnd1 and zip1 mutants, respectively.

Fig 4. Rad51 and Dmc1 sr foci are extremely small and clustered at sub-diffraction distances.

(A-D) dSTORM micrographs of Rad51 filaments assembled on a linear 2.7 kbp dsDNA in vitro in the presence of the meiotic protein Hed1. Corresponding widefield micrographs are inset at top right and the scale bars are 1 μm wide (E-H) Example dSTORM and widefield (bottom right insets) micrographs of wild type nuclei stained for (E) Rad51 and (F) for Dmc1; mnd1 nuclei stained for (G) Rad51 and (H) Dmc1. Arrowheads indicate examples of 100 nm paired Rad51 sr foci, magnified in the upper right insets. Scale bar is 1 μm for dSTORM and widefield micrographs, 100 nm in top right insets. (I-K) Distributions of Rad51→Rad51 sr foci nearest neighbor measurements for (I) wild type, (J) mnd1, and (K) zip1 mutants. Micrographs from cultures 3.5 hours after meiotic induction. (L-O) Each Rad51 focus in a pair of Rad51 foci imaged under widefield can contain more than one Rad51 sr focus. (L-N) Micrographs of paired Rad51 foci observed at widefield resolution (left) and with dSTORM (right) containing 1 and 1 (L); 1 and 2 (M); and 2 and 2 (N) Rad51 sr foci. Scale bar is 400 nm. (O) Distribution of the frequencies of Rad51 sr foci in one focus (blue) or both foci (red) in paired Rad51 foci at low resolution. 48 widefield pairs of Rad51 foci were scored. (P) Characterization of the dimensions of Rad51 and Dmc1 sr foci. An ellipse was fit to each sr focus. L_major is the length of the ellipse’s long axis; aspect ratio equals L_major/L_minor. Scoring of 432, 212, and 304 sr foci from 5 wild type, 5 mnd1, and 5 zip1 nuclei for Rad51 scoring; 359 and 592 sr foci from 3 wild type and 6 mnd1 nuclei for Dmc1 scoring. Errors are S.D.

Fig 5. Elongated Dmc1 structures and higher order clustered Rad51 structures accumulate in strand exchange mutants at late times.

(A-D) 8 hr mnd1 nucleus stained for Dmc1. (A) dSTORM and (B) widefield micrographs each with 1 μm scale bars. (C) Magnified region from (A) indicated by arrowhead, highlighting elongated Dmc1 structure. Scale bar is 100 nm wide. (D) Surface plot of (C). (E-H) 3.5 hr dmc1 nucleus stained for Rad51, highlighting a common pair (low order cluster) of sr foci. (I-L) 8 hr mnd1 nucleus and (M-P) 8 hr dmc1 nucleus stained for Rad51, highlighting higher order clusters of Rad51 sr foci. (Q-T) dSTORM micrographs of elongated Rad51 structures observed in spread nuclei of mitotic srs2 mutants overexpressing Rad51. Scale bar is 1 μm.

Fig 6. Recombinosome Model: Rad51 and Dmc1 each form short filaments on both spatially separated ends of a DSB.

(Top) Short helical Rad51 and Dmc1 nucleoprotein homofilaments (green and red, respectively) form on adjacent segments of a single DSB-associated ssDNA tract. Each of these filaments is on the order of 100 nt or 33 protomers long and more than one Rad51-Dmc1 structure can form on a single tract of ssDNA. This single end of the DSB manifests itself cytologically as a side-by-side Rad51-Dmc1 co-focus (represented by the offset transparent red and green circles). (Left) Both Rad51 and Dmc1 similarly occupy the second end of the DSB. Prior to strand exchange, the two ends of the DSB are separated by 400 nm or less, resulting in focus pairing. The two ends of the DSB are both tethered to the axial element (purple box) by chromatin arms of variable length. Sister chromatids (pink circles) are often split in a DSB-independent manner. (Right) This architecture is maintained after strand exchange tethers the homolog at the predetermined distance of 400 nm or less.