Caenorhabditis elegans DSB-3 reveals conservation and divergence among protein complexes promoting meiotic double-strand breaks

Abstract

Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: Recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved. Here, we identify DSB-3 as a DSB-promoting protein in the nematode Caenorhabditis elegans. Mutants lacking DSB-3 are proficient for homolog pairing and synapsis but fail to form crossovers. Lack of crossovers in dsb-3 mutants reflects a requirement for DSB-3 in meiotic DSB formation. DSB-3 concentrates in meiotic nuclei with timing similar to DSB-1 and DSB-2 (predicted homologs of yeast/mammalian Rec114/REC114), and DSB-1, DSB-2, and DSB-3 are interdependent for this localization. Bioinformatics analysis and interactions among the DSB proteins support the identity of DSB-3 as a homolog of MEI4 in conserved DSB-promoting complexes. This identification is reinforced by colocalization of pairwise combinations of DSB-1, DSB-2, and DSB-3 foci in structured illumination microscopy images of spread nuclei. However, unlike yeast Rec114, DSB-1 can interact directly with SPO-11, and in contrast to mouse REC114 and MEI4, DSB-1, DSB-2, and DSB-3 are not concentrated predominantly at meiotic chromosome axes. We speculate that variations in the meiotic program that have coevolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery.

Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity

“…In the predicted DSB-1:2:3 trimer, the alpha-helical C-termini of DSB-1 and DSB-2 wrap around each other to form a channel which accommodates the helical N-terminus of DSB-3; a similar structure was predicted for a trimer of two Rec114 and one Mei4 ( Supplemental Figure 5A ). In all species (nematode, yeast, and human), these structural predictions are in agreement with previous models based on yeast two-hybrid (25, 63) and crosslinking mass spectrometry analysis (26). This trimer prediction was not found in models of a DSB-2:DSB-2:DSB-3 trimer, but was found in three out of five DSB-1:DSB-1:DSB-3 models.…”

“…These models raise the possibility that DSB-1 dimers are more likely to bind DSB-3 in the absence of DSB-2, than DSB-2 dimers are to bind DSB-3 in the absence of DSB-1. This asymmetry would be consistent with the more severe phenotype of dsb-1 compared to dsb-2 mutants, as well as with yeast two-hybrid evidence showing DSB-1 but not DSB-2 directly binds to DSB-3 (25). The consistency of the structural prediction in three highly-diverged species, and its agreement with known in vivo data, is highly suggestive of a conserved interaction.…”

“…The recent identification of dsb-3 as a nematode ortholog of Mei4, and its likely participation in a complex with DSB-1 and DSB-2 (25) akin to the heterotrimeric Rec114-Mei4 complex of yeast (26) prompted us to examine predicted structural properties of such a complex using the AlphaFold structure prediction pipeline (61, 62). Since both DSB-1 and DSB-2 are orthologs of Rec114, we tested three possible complexes: a fully heterotrimeric complex of DSB-1, DSB-2, and DSB-3, and complexes containing two copies of either DSB-1 or DSB-2 with one copy of DSB-3.…”

“…Homologs of yeast Rec114 include mouse Rec114 (20, 21), fission yeast Rec7 (22), and C. elegans DSB-1 and DSB-2, all of which are required for meiotic DSB formation (23, 24). A homolog of Mei4, DSB-3 in C. elegans , is also required for DSB formation (25), but no nematode homolog of Mer2 has been identified as of this writing. While the exact mechanism of DSB promotion by the RMM complex remains obscure, recent evidence suggests it may act as a scaffold for the Spo11 core complex (26).…”

Evolution and diversity of the TopoVI and TopoVI-like subunits with extensive divergence of the TOPOVIBL subunit

“…In Placozoa and C. elegans , alternative strategies may have been selected, for instance by involving other partners for binding to SPO11. Interestingly, while the placozoan T. adhaerens has a single REC114 homolog, C. elegans has two REC114 paralogs (DSB-1 and DSB-2) of which one (DSB-1) interacts with SPO11-1 in Y2H assays (Hinman et al, 2021). This suggests that REC114 function is maintained, but acts by interacting with SPO11 rather than with TOPOVIBL.…”