C. elegansDSB-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 for 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 meiotic 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 co-evolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery.

Structure and DNA-bridging activity of the essential Rec114–Mei4 trimer interface

“…Also, the poor prediction confidence of the DSB-3 SSM2 region (pLDDT score in Supplemental Fig. S6C) is consistent with this segment being less well conserved (Hinman et al 2021).…”

“…Interfacial residues-particularly hydrophobic ones that contribute to both homotypic and heterotypic intermolecular interactions-are the principal contributors to the signatures of sequence conservation in these parts of both proteins. Our findings thus provide a fine-grained molecular framework for understanding the coevolution of these proteins, explaining in turn how they often act as a single functional unit during meiotic DSB formation (Li et al 2006;Maleki et al 2007;Kumar et al 2018;Hinman et al 2021;Vrielynck et al 2021). Nonetheless, it is remarkable that many residues in this domain are highly variable between species.…”

“…Meiotic recombination starts with DNA double-strand breaks (DSBs) formed by Spo11 protein (related to archaeal topoisomerase VI) plus a cohort of additional conserved factors (Keeney 2008;Robert et al 2016). Among these factors are Rec114 and Mei4, which are essential for DSB formation but also regulate the number, timing, and location of DSBs in many species (Henderson et al 2006;Carballo et al 2013;Rosu et al 2013;Stamper et al 2013;Murakami and Keeney 2014;Kumar et al 2010Kumar et al , 2018Papanikos et al 2019;Boekhout et al 2019;Mu et al 2020;Claeys Bouuaert et al 2021a;Hinman et al 2021).…”

“…Rec114 and Mei4 from Saccharomyces cerevisiae form a 2:1 heterotrimeric complex in vitro and assemble cooperatively on DNA to form dynamic nucleoprotein conden-sates (Claeys Bouuaert et al 2021a;Yadav and Claeys Bouuaert 2021). In vivo, they associate early in meiotic prophase I with chromatin and form colocalized and interdependent foci along chromosome axes in multiple species (Li et al 2006;Maleki et al 2007;Kumar et al 2010Kumar et al , 2018Panizza et al 2011;Rosu et al 2013;Stamper et al 2013;Boekhout et al 2019;Papanikos et al 2019;Hinman et al 2021). Rec114-Mei4 (RM) complexes from different organisms interact directly with the meiotic TopoVI-like complex (Arora et al 2004;Maleki et al 2007;Claeys Bouuaert et al 2021a;Hinman et al 2021;Vrielynck et al 2021;Nore et al 2022), but the molecular mechanisms of RM function remain poorly understood.…”