Meiotic DNA break resection and recombination rely on chromatin remodeler Fun30

DNA double-strand breaks (DSBs) are nucleolytically processed to generate single-stranded DNA tails for homologous recombination. In Saccharomyces cerevisiae meiosis, this 5’-to-3’ resection involves initial nicking by the Mre11–Rad50–Xrs2 complex (MRX) plus Sae2, then exonucleolytic digestion by Exo1. Chromatin remodeling adjacent to meiotic DSBs is thought to be necessary for resection, but the relevant remodeling activity was unknown. Here we show that the SWI/SNF-like ATPase Fun30 plays a major, non-redundant role in resecting meiotic DSBs. A fun30 null mutation shortened resection tract lengths almost as severely as an exo1-nd (nuclease-dead) mutation, and resection was further shortened in the fun30 exo1-nd double mutant. Fun30 associates with chromatin in response to meiotic DSBs, and the constitutive positioning of nucleosomes governs resection endpoint locations in the absence of Fun30. We infer that Fun30 directly promotes both the MRX- and Exo1-dependent steps in resection, possibly by removing nucleosomes from broken chromatids. Moreover, we found that the extremely short resection in the fun30 exo1-nd double mutant is accompanied by compromised interhomolog recombination bias, leading to defects in recombination and chromosome segregation. Thus, this study also provides insight about the minimal resection lengths needed for robust recombination.

(D) ARS and tRNAs that are closer to Rec114 binding sites tend to exhibit higher DSB-dependent Fun30 ChIP-seq signals.The ARS and tRNA regions from panels B and C were subdivided into two groups based on the distance to the nearest Rec114 peak or hotspot center: "close" indicates elements less than the median distance away and "far" indicates the rest.Proximity to Rec114 peaks was associated with a significantly higher DSB-dependent Fun30 ChIP-seq signal, whereas proximity to hotspots showed no such pattern.These results suggest that at least some of the DSB-dependent recruitment of Fun30 to ARS or tRNA genes is a consequence of fortuitous proximity or overlap of (some of) these elements with Rec114 ChIP peaks.

Figure S1 .
Figure S1.S1-seq reproducibility and S1-seq in swr1∆ and rad9∆ (A) Reproducibility of S1-seq between two biological replicates of each genotype.Each point is the log2transformed read count in a 1-kb segment of the genome.(B) Preservation of hotspot heats in fun30∆.Each dot represents the sum of the S1-seq signal at a given hotspot in a sae2∆ background (n = 3908).S1-seq library preparation was previously shown to quantitatively capture the unresected DSBs that accumulate in sae2∆ mutants (Mimitou et al. 2017; Mimitou and Keeney 2018).(C) Preservation of fine-scale DSB distributions within hotspots in fun30∆.For each of 3908 DSB hotspots, we computed the correlation coefficient (Pearson's r) comparing the S1-seq spatial distribution between the indicated pairs of datasets, then plotted the distributions of the 3908 correlation coefficients.The vertical dashed lines indicate the mean r value for each comparison.We compared two biological replicates of sae2∆ to show the intrinsic variability in this measurement (mean r = 0.66) and we compared sae2∆ to sae2∆ spo11-F260A (Claeys Bouuaert et al. 2021) as an example of what happens in a mutant with altered DSB distributions (mean r = 0.36).Comparison of sae2∆ with fun30∆ sae2∆ gave a distribution of correlation coefficients indistinguishable from the comparison of sae2∆ replicates (mean r = 0.67), indicating that the fun30∆ mutation has little or no measurable effect on local DSB distributions within hotspots.(D) Reproducibility of swr1∆ and rad9∆ S1-seq biological replicates.(E) Average S1-seq distribution around hotspots in swr1∆ and rad9∆.(F) Distribution of resection tract lengths in swr1∆ and rad9∆ as in Figure 2C.

Figure S2 .
Figure S2.DSB-dependent Fun30 enrichment (A) Total Fun30 ChIP-seq coverage normalized to the spike-in control.Bars are the means from two biological replicates; open circles show the individual values for each replicate.(B) Average Fun30 ChIP-seq signals around ARS, tRNA, and centromere.The random sites here and in panel C are the same as in Figure 3B.(C) DSB-dependent Fun30 enrichment.Box plots summarize the distributions across all of the indicated elements from panel B and Figure 3B for Fun30 ChIP-seq signal summed in 1-kb windows.Note the different y-axis scales for left and right parts of the plot.In all box plots, thick horizontal bars denote medians, box edges mark the upper and lower quartiles, and whiskers indicate values within 1.5-fold of the interquartile range.Outliers are not shown.Here and in panel D, numbers above brackets indicate P values of two-sided Wilcoxon tests.(D)ARS and tRNAs that are closer to Rec114 binding sites tend to exhibit higher DSB-dependent Fun30 ChIP-seq signals.The ARS and tRNA regions from panels B and C were subdivided into two groups based on the distance to the nearest Rec114 peak or hotspot center: "close" indicates elements less than the median distance away and "far" indicates the rest.Proximity to Rec114 peaks was associated with a significantly higher DSB-dependent Fun30 ChIP-seq signal, whereas proximity to hotspots showed no such pattern.These results suggest that at least some of the DSB-dependent recruitment of Fun30 to ARS or tRNA genes is a consequence of fortuitous proximity or overlap of (some of) these elements with Rec114 ChIP peaks.

Figure S3 .
Figure S3.MRX/Sae2 nicks within the +1 nucleosome Examples of resection endpoint distributions in exo1-nd fun30∆ at three representative loci that contributed to the average shown in Figure 4B.S1-seq signals (41-bp smoothed) from the top (blue) or bottom (red) strand are shown, dependent on the orientation of the gene where the +1 nucleosome is located.Numbers in light blue indicate the percentages of S1-seq signal in the four windows spanning the +1 nucleosome and NDR (see legend to Figure 4B).

Figure S4 .
Figure S4.Physical assay detecting recombination intermediates at the HIS4LEU2 hotspot (A) Physical map of the HIS4LEU2 locus showing diagnostic XhoI restriction enzyme sites and the position of Southern blot probe A. "Mom" and "Dad" indicate the two parental versions of the locus; COs, crossovers; NCOs, non-crossovers; MM' IS-dHJ, intersister double-Holliday junction; MD IH-dHJ, interhomolog double-Holliday junction; DD' IS-dHJ, intersister double-Holliday junction; SEIs, single-end invasions.Positions of XhoI sites are indicated as circled Xs. (B) Example one-dimensional gel analysis showing parental signals, DSBs, COs, NCOs, and joint molecules (JMs).The Southern blot image is reproduced from Figure 6A.(C) Example two-dimensional gel displaying parental signals and recombination intermediates.Green arrow or text indicate interhomolog species; red and blue arrows and text indicate intersister species.(D) Key steps in crossover and noncrossover formation during meiosis.(E) Representative gel images of crossovers and noncrossovers.(F) Quantification of crossovers and noncrossovers (mean ± SD for three independent meiotic cultures).

Figure S5 .
Figure S5.Analysis of early meiotic progression by flow cytometric DNA analysis.(A) Representative histograms of flow cytometric measurements of the cellular DNA content.(B) Progression of pre-meiotic DNA replication during early meiosis.The percentage of cells in G2 phase was calculated from flow cytometry (mean ± SD for three independent meiotic cultures).

Figure S6 .
Figure S6.DSBs formation at various natural hotspots.(A-H) Representative one-dimensional gel analyses of DSBs and corresponding quantification at the ERG1 (A and B), CYS3 (C and D), BUD23 (E and F), and ARG4 (G and H) hotspots.Error bars indicate mean ± SD for three independent cultures.

Figure S7 .
Figure S7.Schematic representation of DSB-dependent Fun30 recruitment in the tethered loop-axis complex A model proposed based on findings in this study.In response to Spo11 cleaving DNA within the TALC, Fun30 is recruited to the DSB ends and remodels the nucleosomes.

Table S2 . Genetic distance and MI nondisjunction estimated by fluorescent spore assay
THR1 interval was omitted as they are indistinguishable with MI-NDJ(Thacker et al. 2011).Wild type data are from our previous publication(Thacker et al. 2011).p values for genetic distance were calculated by G test.