DNA-dependent macromolecular condensation drives self-assembly of the meiotic DNA break machinery

Abstract

Formation of meiotic DNA double-strand breaks (DSBs) by Spo11is tightly regulated and tied to chromosome structure, but the higher-order assemblies that execute and control DNA breakage are poorly understood. We address this question through molecular characterization of Saccharomyces cerevisiae RMM proteins (Rec114, Mei4 and Mer2)-essential, conserved components of the DSB machinery. Each subcomplex of Rec114-Mei4 (2:1 heterotrimer) or Mer2 (homotetrameric coiled coil) is monodisperse in solution, but they independently condense with DNA into dynamic, reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive condensation, which correlates with DSB formation in vivo. Condensates fuse into mixed Rec114-Mei4-Mer2 clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centers of DSB activity. We propose that multilayered control of Spo11 arises from recruitment of regulatory components and modulation of biophysical properties of the condensates.The role of Rec114-Mei4 and Mer2 in meiosis

Novel mechanistic insights into the role of Mer2 as the keystone of meiotic DNA break formation

“…Furthemore, Spp1 associated with Mer2 has a longer residence time on nucleosomes when compared with Spp1 when part of COMPASS 15 residues 140-256 of Mer2 12,13 which we from now on refer to as Mer2 "core" ( Figure 1B). We measured the molecular mass of Mer2 core by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and concluded that the Mer2 core is a tetramer ( Figure 1D), consistent with recently published data 17 . We also find that while most Mer2 constructs lacking the core are monomeric, there is also a dimerisation region in the C-terminal region between residues 255 and 314 ( Figure 1D).…”

“…Presumably this is essential given that the interaction between COMPASS bound Spp1 and H3K4 me3 is transient, whereas the association of the DSB forming machinery is an apparently more stable event 15 . Given that the interaction domain is the same as the tetramerisation domain we speculate that the antiparallel arrangement of coiled-coils of Mer2 core form two mirrored binding sites for Spp1, with V195 at the centre 14,17 .…”

Structural and functional characterization of the Spo11 core complex

“…This strongly suggests that Spo11 complexes are tethered on a surface (Johnson et al, 2019). Based on the intrinsic property of Rec114-Mei4 and Mer2 to undergo DNA-driven macromolecular condensation (Claeys Bouuaert et al, 2020), we propose that this surface is composed of higher-order Rec114-Mei4-Mer2 nucleoprotein assemblies attached to the chromosome axis (Supplementary Figure 10B). This surface is therefore occupied by multiple Spo11 subunits that can simultaneously engage the DNA substrate.…”

“…Model of assembly of the DSB machinery. DNA-driven condensation by Rec114-Mei4-Mer2 is proposed to provide a platform that recruits the core complex, where it engages its DNA substrate (Claeys Bouuaert et al, 2020). 'Biot' indicates the position of a 3'-biotin modification.…”

Phase Separation during Germline Development

“…In budding yeast, the Spo11 accessory proteins Rec114, Mei4, and Mer2 (RMM) bind stably to the chromosome axis as foci [73][74][75][76][77]. In the presence of DNA, RMM can phase-separate in vitro, and the resulting condensates can recruit Spo11 [78]. RMM mutants with reduced DNA-binding activity form fewer foci and fail to from double-strand breaks in vivo [78].…”