Insights into the recruitment of the H3K4me3 reader Spp1 by the meiotic double-strand break protein Mer2.

The formation of DNA double-strand breaks (DSBs) by Spo11 is tied to the loop-axis organization of meiotic chromosomes. Before DSB formation, chromatin loops marked by histone H3K4 trimethylation become tethered to the chromosome axis through interactions between Spp1 and Mer2. Mer2 is an essential partner of Spo11 thought to assemble the DSB machinery via biomolecular condensation, but these molecular assemblies remain poorly characterized. Here, using AlphaFold modeling, biochemical reconstitution, and biophysical validation, we explored the relationship between Mer2, Spp1, and their DNA-bound complexes. The tetrameric coiled-coil domain of Mer2 has four rotationally symmetrical sites that can bind a C-terminal α-helix of Spp1. However, Mer2-Spp1 complexes assemble with a 4×2 stoichiometry, perhaps through allosteric modulation or steric hindrance. Mer2 also accommodates multiple DNA duplexes, allowing the assembly of tripartite Mer2-Spp1-DNA complexes with branched DNA substrates and effective recruitment of Spp1 within nucleoprotein condensates. However, because the Spp1- and DNA-binding sites of Mer2 partially overlap, Spp1 recruitment reduces DNA binding by Mer2, which is compensated by positively charged residues within Spp1. Nevertheless, this DNA-binding motif appears to be dispensable for the meiotic function of Spp1. These findings provide insights into the structural organization of Mer2 and Spp1 and their role in the assembly of the meiotic DSB machinery.