XND-1 and HIM-17 interact and are predominantly localized on DNA loops.

A) Summary of the yeast two-hybrid assay results. The complete set of results is given in Figure S1A. B) Western blot analysis of endogenous XND-1 (top) and HIM-5::GFP::FLAG (bottom) on HA pull-downs performed in him-17::3xHA; him-5::GFP::3xFLAG strain. Red box indicates the XND-1 band. Analysis was performed in biological duplicates. Specificity of the anti-XND-1 antibody is shown in Figure S3A. C) Quantification of embryonic viability (hatching rates) and frequencies of male offspring among the progeny of the indicated genotypes. Data are shown as mean +/- SD; * p<0.05, **** p < 0.0001, ND= not determined, n= number of P0 parents whose brood was examined. D) Representative confocal images of mid-pachytene stage nuclei showing that juxtaposed XND-1 and HIM-17 localization away from the chromosome axes (stained with anti-HTP-3). The X chromosome is not stained by either XND-1 and HIM-17 (Red arrows). Scale bars = 20μm. E) Quantification of the pairwise overlap between XND-1, HIM-17, and the chromosome axes. F) Alphafold3 model of XND-1-HIM-17 dimer. The black line shows the 180 Å distance between the HA tag in HIM-17 and the XND-1 protein which is recognized by the polyclonal antibody.

DSB-1 associates with HIM-5 and regulates its localization

A) Summary of yeast two-hybrid results with dsb-1, dsb-2, dsb-3 and him-5. The complete set of results is given in Figure S1A. B) Western blot analysis of anti-GFP pull-downs performed in GFP::dsb-1;him-5::3XHA strain showing co-IP of HA-tagged HIM-5 proteins. Analysis was performed in biological duplicates. C) Immunofluorescence analysis of HIM-5 localization in Phim-5::him-5::GFP;him-5 and Phim-5::him-5::GFP;dsb-1 backgrounds. DNA (DAPI, gray), HIM-5::GFP (anti-GFP, green), and chromosome axes (anti-pHTP-3, magenta). pHTP-3 staining delineates entrance to the transition zone (marked dotted yellow lines), while arrows indicate the pre-meiotic localization of HIM-5. D) Quantification of DAPI-stained bodies at diakinesis for the indicated genotypes. Colors correspond to the number of DAPI-stained bodies shown in the key below for N2, dsb-1, and dsb-2 mutants expressing Ppie- 1::him-5::GFP. Sample sizes (N) are indicated. Statistical significance for comparisons between groups is shown at the top (**p < 0.01). E) Quantification of embryonic viability (hatching rates) and frequencies of male offspring among the progeny of the indicated genotypes. Data are shown as mean +/- SD; NS stands for not significant; *** p < 0.001, n= number of P0 parents whose brood was examined.

Genetic and protein-protein interaction studies show evidence of SPO-11 accessory protein sub-complexes in C. elegans.

A) Summary of the genetic interaction results. The complete set of results is given in Figure S5. B) Genetic groupings based on data presented here and previously published (Chung et al., 2015; Mateo et al., 2016; McClendon et al., 2016; Janisiw et al., 2020). C) Summary of yeast two-hybrid results. The complete set of results are shown in Figure S1A. D) Schematic representation of the interaction network based on Y2H interactions. Thick line: Strong interaction; thin line: weak interaction. E, F) Yeast-two-hybrid interactions of SPO-11, DSB-2, and DSB-3 with different DSB-1 sub-domains (schematic in F). Details on DSB-1 sequence, protein structure, and deletions used in these experiments, are found in Figure S8. G) AlphaFold2 predictions of DSB-1, DSB-2 and DSB-3 protein structures. H, I) Predicted interaction domains of DSB-1, DSB-2 with DSB-3 based on homology with the yeast proteins. The DSB-1/-1/-3 trimer is thermodynamically more stable than the DSB-1/-2/-3 trimer, consistent with the subordinate role for DSB-2 in break induction in young animals.

Speculative model of the DSB formation complex in C. elegans

Synthesizing prior work and data herein, we propose a model to explain the known interactions and localization patterns of the DSB regulatory factors in C. elegans. DSB-1/-2/-3 and HIM-17-XND-1 subcomplexes are initially found on DNA loops. The phosphorylation of REC-1 by CDK activates DSB formation, allowing HIM-5 through its association with MRE-11 and PARG-1 (which both associate with HTP-3) to efficiently bring the DSB-1/-2/-3 complex with SPO-11 to the chromosome axis. This recruitment to the axes by HIM-5, facilitates the coupling of DSBs to downstream repair by the MRN complex and others. In the absence of HIM-5, DSBs/COs on autosomes do not occur efficiently and crossover sites are shifted to the gene-rich third of each chromosome, presumably because DSB-1/-2/-3/SPO-11 cannot be as efficiently recruited to the proper sites partially defined by HIM-17 and XND-1. We assume that recruitment to the chromosome axes in the absence of HIM-5 is likely stochastic or mediated by as yet unknown protein interactions. On the X chromosome, the absence of HIM-5 prevents the formation of most DSBs.

Yeast-2-Hybrid studies and control IP data.

A) Representative Y2H interactions between DSB factors, as monitored by growth in different media: (left) -L-W used as a positive control; (center) -L-W-H showing weak interactions and, in some cases, self-activation; (right) -L-W -Ade showing strong interactions and self-activation by CEP-1. Every DSB factor was tested here as activating domain (AD) fusions as well as reciprocal DNA binding domain (BD) fusions. B) Western blot showing immunoprecipitation of HIM-17 from him-17::GFP whole worm lysates using anti-GFP antibody. Unbound protein fraction (flow through, F) and proteins bound to the GFP trap beads (B) from wild type and him-17::GFP transgenic worm lysates are shown. The HIM-17::GFP-specific signal is enriched by using a GFP trap. C) Western blot showing immunoprecipitation of HIM-17 from him-17::3xHA;him-5::GFP::3xFLAG whole worm lysates using an HA affinity matrix.

Quantification of DAPI-stained bodies at diakinesis in him-17(ok424) shows lack of rescue by HIM-5 expressed from its endogenous promoter.

Colors correspond to the number of DAPI-stained bodies shown in the key. No statistical difference is observed.

Localization of XND-1 and HIM-17 are non-overlapping and not interdependent.

A) Western blot showing specificity of the guinea-pig anti-XND-1 antibody. Western analysis was performed on protein extracts from wild-type N2 worms and two independent xnd-1 mutant strains. A distinct band corresponding to XND-1 (red square) is detected only in the N2 extract, confirming the specificity of the antibody. No signal is observed in the mutant strains, consistent with loss of xnd-1 expression (see also Wagner et al., 2010). B) 3xHA::HIM-17 and anti-XND-1 staining do not over overlap with one another or with the DNA axes. Shown here are 3D renderings of confocal stacks from the mitotic zone, early-middle pachytene, and mid-late pachytene regions. C) Localization of XND-1 is normal in him-17(ok424M-Z-) mutants (anti-XND-1, pink, DNA/DAPI, green) (top). Localization of 3xHA::HIM-17 is unaffected in xnd-1(ok709) mutants (anti-HA, pink; DNA/DAPI, green) (bottom).

Localization of endogenous HIM-5 in dsb-1 mutants.

A) Immunofluorescence analysis of HIM-5::HA in dsb-1 mutants. DAPI (blue) stains DNA, anti-HA marks endogenously tagged HIM-5 (green), and HTP-3 (red) labels chromosome axes. HIM-5 appears localized in nuclei in pre-meiotic stages (indicated by arrows). However, after the transition zone (TZ), HIM-5 loses its nuclear localization. B) Top: C. elegans gonad fixed and stained with DAPI to show the organization and distribution of the nuclei along the Prophase I. Bottom: live imaging of nuclei in the transition zone (leptotene-zygotene) and middle-pachytene. eaIs15 (Ppie-1::him-5::GFP) is visualized in freshly dissected gonads by GFP fluorescence (green), and DNA by DRAQ5 (red). In dsb-1 mutants, HIM-5 is nuclear in the transition zone and then only appears in cytoplasmic puncta by middle pachytene.

Epistasis analysis of DSB factors defines multiple genetic groups for crossover formation.

A-N) Crossover formation is assessed by the number of DAPI-staining bodies at diakinesis. Each graph shows the quantification of DAPI-bodies in diakinesis nuclei for the indicated single and double mutants. Color indicates the number of DAPI-staining bodies. Sample sizes (N) are indicated. Statistical significance for comparisons between groups is shown at the top (NS= not significant, **p < 0.01, *** p<0.001, **** p< 0.0001).

Mixed phenotypes are seen in cep-1(lg12501); mre-11(iow1) double mutants.

A and E) Quantification of the number of DAPI-stained bodies at diakinesis for the indicated genotypes. Color indicates the number of DAPI-stained bodies. B-D) Representative DAPI-stained images of oocytes in diakinesis for cep-1;mre-11 worms showing univalents (B, C) and fusions (D).

Irradiation rescues crossover defects of accessory factor double mutant strains.

Quantification of DAPI-stained bodies at diakinesis for indicated genotypes with and without 10Gy of γ-irradiation. Color indicates the number of DAPI-staining bodies. Sample sizes (N) are indicated. Statistical significance for comparisons between groups is shown at the top (**** p< 0.0001).

Structural analysis of DSB-1 identified potential interaction motifs.

A) DSB-1 protein sequence with secondary structures demarcated: beta-sheets (blue); alpha-helices (purple). Deletions for Y2H assay shown in Figure 3 are marked with red and highlight in B) Alphafold2 model of DSB-1: helix 2 (left); helices 3-6 (right).

Strains and genetics.

All strains were derived from the wild-type Bristol strain N2 and were cultivated at 20 °C under standard conditions. Abbreviated names and full genotypes of the strains used in this study are listed here.

Immunoprecipitation and Mass Spectrometry (IP-MS) Results of HIM-17::GFP from whole worm extracts.

Sequence coverage refers to the percentage of the protein sequence that was pulled down in the HIM-17::GFP IP samples. Numbers are from two independent biological repeats and represent specific enrichment in HIM-17::GFP compared to control IPs (see Methods).