Meiotic crossover control by concerted action of Rad51-Dmc1 in homolog template bias and robust homeostatic regulation

Abstract

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1's strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51's strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1's chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1's dominance in promoting strand exchange between homologs involves repression of Rad51's strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.

Figure 1. Analysis of DSBs and crossovers in hed1 and dmc1 hed1 mutants.

A. Map of the HIS4::LEU2 hotspot showing the DSB site, XhoI restriction sites (circled Xs) and the position of the probe used in Southern analysis. Sizes of diagnostic fragments are shown below. B. 1D Southern analysis of DSBs and crossovers at HIS4::LEU2. Time points are 0, 2, 3, 3.5, 4, 4.5, 5, 6, 7, 9, and 11 hours. Asterisk highlights bands diagnostic of non-allelic recombination between HIS4::LEU2 and leu2::hisG located ∼25 kb away at the native LEU2 locus. C. Quantitation of images shown in (B). % DNA is percentage of total hybridizing DNA signal. MI±MII is the percentage of cells that have completed one or both meiotic divisions. The rightmost panel shows quantification of Rad51 immuno-staining foci in spread nuclei. Each data point shows average Rad51 focus counts per nucleus (n = 50). The later data are from different time courses to those analyzed in the first three graphs.

Figure 2. hed1 and dmc1 hed1 mutants are defective for interhomolog bias at two recombination hotspots.

A. Diagram of JM structures detected at the HIS4::LEU2 hotspot, from lowest to highest mobility. Positions of diagnostic XhoI sites (circled Xs) and the Southern probe are also highlighted. B. Images of 2D gels for time points that represent the peaks of JM abundance. Lower panels show magnifications of the JM signals. The positions of dHJs, SEIs, and mcJMs (multi-chromatid JMs) are indicated. C. Quantitation of JMs from 2D analysis. IH/IS ratio is the ratio of IH-dHJs/IS-dHJs. See Figure S1 for independent analysis of the same set of strains. D. Diagram of JM structures detected at the ERG1 hotspot. Positions of SacII sites (circled Ss) and the probe are shown. E. Images of 2D analysis at ERG1 in wild type and dmc1 hed1 strains. Positions of IH- and IS-dHJs are indicated. F. IH/IS dHJ ratios at ERG1 in wild type and dmc1 hed1 strains. G. 1D Southern analysis of crossing-over at ERG1 in wild type and dmc1 hed1 strains. H. Quantitation of images shown in (G). Crossover levels are shown for the 24 hr time point.

Figure 3. Mnd1 promotes Dmc1-dependent interhomolog recombination and Dmc1 inhibits Rad51-mediated recombination.

A. Images of 1D Southern analysis at HIS4::LEU2 for wild-type, dmc1, mnd1, mnd1 hed1, dmc1 hed1, and mnd1 dmc1 hed1 time-course experiments. B. Quantification of the first meiotic division (MI±MII), DSBs, and COs for wild-type, dmc1, mnd1, mnd1 hed1, dmc1 hed1, and mnd1 dmc1 hed1 time-course experiments. C. Images of representative panels from 2D Southern analysis with corresponding enlargements of the JM regions. D. Quantitation of JMs from 2D Southern analysis.

Figure 4. Mek1 and Hed1 make independent contributions to interhomolog template bias.

A. Images of representative panels from 2D Southern analysis, with corresponding enlargements of the JM regions. B. Analysis of the first meiotic division in the indicated strains. C. Quantification of JM formation and IH/IS dHJ ratios in the indicated strains. Note that the signals trailing horizontally to the left of the parental species (“Mom” and “Dad”) results from overloading of the lanes. Samples were overloaded to aid detection of faint JM species. See Figure 1B for position of parental species.

Figure 5. Synaptonemal complex formation is defective in hed1 and dmc1 hed1 strains.

A. Representative image of a spread meiotic nucleus from the dmc1 hed1 strain showing greater than 16 Ctf19 foci: immunostain for Zip1 is shown in green, Ctf19 stain is shown in red. Colocalization of a Ctf19 focus with an elongated Zip1 structure is indicated by an arrow. B. Quantitation of the percentage of prophase (Zip1 positive) nuclei displaying more than 16 Ctf19 foci as an indicator of the fraction of prophase nuclei containing one of more unpaired chromosomes, i.e. nuclei displaying “incomplete pairing.” Nuclei with fewer than 14 Ctf19 foci, which represented less than 15% of total Zip1⁺ nuclei, were excluded from the analysis. Approximately 50 nuclei were scored for each time point. The experiment was carried out 3 times; means and standard deviations are shown. Asterisks indicate that >95% of wild type and hed1 single mutant cells had exited prophase by 10 hrs (most with evidence for insipient spore formation); these samples were not scored for Ctf19 foci. C. Representative images of spread nuclei immunostained for Zip1 in green and Red1 in red. Arrows indicate Zip1 polycomplexes (PC). The dmc1 micrographs show a Class I nucleus (Zip1 puncta only) with a PC (white arrow); the dmc1 hed1 nucleus is class II (Zip1 puncta and lines); and the wild-type and hed1 nuclei are class III (predominantly linear Zip1 structures). D. Quantification of Zip1 staining classes over time. 50 nuclei were analyzed for each time point. E. Analysis of staining intensities of Red1and Zip1 in regions containing elongated Zip1 structures. Units are arbitrary. Quantitation can be found in Table S1. F. Representative images of individual spread chromosomes immunostained for Zip1 in green and Red1 in red from both wide-field and STED microscopy. Images are cropped and pasted on a black background. Note differences in the staining intensity of Red1 relative to Zip1. G. STED images of Red1 immunostaining (red). Corresponding Zip1 staining (green) was imaged at lower resolution via confocal microscopy. The top image pair is a wild-type nucleus stained with both Red1 and Rec8 primary antibodies and a secondary antibody that recognized both proteins shown in red. In the other 3 image pairs, the red signal is from anti-Red1 alone; no anti-Rec8 was used. Regions showing parallel linear rows of punctate staining for Rec8+Red1 (or Red1 alone) are indicated with arrows. Additional STED images are provided in Figure S5. Quantitation can be found in Table S2.

Figure 6. Analysis of crossing-over in hed1 and dmc1 hed1 mutants.

A. Map of marker configurations along chromosome III. Note that this strain does not carry the artificial HIS4::LEU2 hotspot. B. Map distances for 8 genetic intervals along chromosome III. Map distances were calculated via the method of Perkins (T+3NPD/total ×100). Sample sizes were 1039, 487, and 1538 tetrads with four viable spores for wild type, hed1, and dmc1 hed1 respectively. Error bars represent standard error. C. Cumulative chromosome III map distances. D. Distribution of tetrads with various numbers of viable spores. Spore viabilities are 96% (4535/4716) in wild type, 96% in (2106/2184) hed1, and 70% (8523/12208) in dmc1 hed1. E. Distribution of chromosome III crossover classes. The red box highlights E0 tetrads that lack crossovers between chromosome III. F. NPD ratios for 6 intervals along chromosome III. For this analysis, the CHA1-TRP1 and TRP1-HIS4 intervals were combined. Error bars represents standard error. See Table S3 for NPD ratios and statistical tests. Asterisks mark intervals in which the reduction in the strength of interference relative to that in wild-type cells is significant. G. Map distance ratios (Adj^CO/Adj^PD) for adjacent intervals. Numbers shown are averages of the two Adj^CO/Adj^PD ratios derived for reciprocal analysis of each adjacent interval pair. Solid lines indicate adjacent intervals for which significant positive interference was detected; dashed lines indicate that the ratio is not significantly different from 1, i.e. no interference. Asterisks mark interval pairs for which the reduction in the strength of interference relative to that in wild-type cells is significant. See Table S4 for map distances and statistical testing.

Figure 7. dmc1 hed1 strains have elevated crossover/non-crossover ratios at two recombination hotspots.

A. Map of the HIS4::LEU2 DSB site showing diagnostic restriction site heterologies. Positions of XhoI (circled Xs), BamHI and NgoMIV sites are indicated. B. 1D Southern analysis of XhoI+NgoMIV doubly digested genomic DNA isolated at 0 and 24 hrs after induction of meiosis. The magnified image highlights bands that are specifically diagnostic of crossovers and noncrossovers. C. Crossover/non-crossover ratios at HIS4::LEU2 calculated from quantification of four independent sets of time courses. Error bars indicate standard error. D. Map of the ERG1 DSB site showing diagnostic restriction site heterologies. Positions of SacII (circled Ss), SalI and SpeI sites are indicated. E. Southern analysis of SacII+SalI doubly digested genomic DNA isolated at 0 and 24 hrs after induction of meiosis. The magnified image highlights bands that are specifically diagnostic of crossovers and noncrossovers. F. Crossover/non-crossover ratios at ERG1 calculated from quantification of four independent sets of time courses. Error bars indicate standard error.

Figure 8. Crossover/noncrossover ratios at ARG4 measured by random spore analysis.

A. Map of the ARG4 region (top) and heterozygous markers used to select ARG4⁺ gene conversions and determine whether they are associated with crossing over (bottom). B. Percent of ARG4 gene conversions associated with crossing over. C. Percentage of viable spores for the strains analyzed in (B).

Figure 9. Crossover homeostasis as a function of interval size.

A. Efficiency of meiotic divisions in parallel cultures of wild-type and dmc1 hed1 cells. B. Physical map of HIS4::LEU2 and adjacent regions of chromosome III showing intervals monitored for crossing-over. Crossovers in interval 1 were monitored by Southern analysis (as in Figure 1A). Crossovers in intervals 2 and 3 were monitored by tetrad analysis. C. 1D Southern analysis of crossing-over in parallel cultures (analyzed in A) of wild-type and dmc1 hed1 cells. Asterisk highlights bands diagnostic of non-allelic recombination between HIS4::LEU2 and leu2::hisG located ∼25 kb away at the native LEU2 locus. D. Quantification of the Southern image shown in (C). Crossover levels at 48 hrs are shown. E. Map distances for intervals 2 and 3 calculated from tetrads sampled from the same cultures analyzed in A, C and D.

Figure 10. Models for Rad51-Dmc1 mediated interhomolog bias and homeostatic responses to inefficient interhomolog interactions.

A. Rad51-Dmc1 dependent interhomolog recombination during meiosis. DNA intermediates leading to interhomolog or intersister recombination are shown. Intersister strand-exchange is presumed to produce intermediates similar to those shown for interhomolog recombination. The 5-fold interhomolog template bias detected in wild-type cells is achieved by the illustrated interactions, as follows: (1) Mek1-Red1 (and presumably Hop1) function together with Rad51 filaments (2) to facilitate filament assembly and interhomolog strand-exchange activity of Dmc1. The grey double arrow indicates interdependence between these two ensembles. (3) Hed1 inhibits Rad51's strand exchange activity and may also influence the stability of Rad51 filaments . However, in vivo, Hed1 is not the major inhibitor of Rad51-mediated strand-exchange, which is largely inhibited by Dmc1 (4). Moreover, Hed1 is not required for the Dmc1-accessory role of Rad51 (2). Hed1 also limits the loading of Red1 onto homolog axes (5). Conversely, Dmc1 promotes Red1 loading (6) suggesting a positive feedback loop between Mek1-Red1 and Dmc1. Finally, Hop2-Mnd1 is essential for Dmc1-mediated interhomolog strand-exchange (7). B. Homeostatic responses compensate for inefficient interhomolog bias. Homologs are represented by thick black lines. Top row: efficient interhomolog bias results in stable pairing and SC formation ensues. Coincident designation of crossover sites and associated interference results in the normal number and distribution of crossovers. If interhomolog bias is inefficient, as seen in dmc1 hed1 cells, pairing and synapsis are delayed and inefficient. Persistent checkpoint signaling resulting from delays in both synapsis and DSB-repair provoke secondary DSB formation and additional interhomolog interactions until stable homolog pairing and synapsis are achieved. Stochastic failure of this process is responsible for crossover failures (non-exchange tetrads). Although the final number of interhomolog interactions is lower than normal, biased outcome results in normal crossover frequencies.