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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 18/01/2026

Finally, we found that Rec102 also interacts with Ski8 across the dimerization plane. Mutation of an interface residue reduces Rec102-Ski8 interaction, dimerization of the core complex on DNA, and meiotic DSB formation.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 18/01/2026

We found that this triple arginine motif is essential for DNA binding by the Spo11 core complex, and it turned out that this patch indeed directly contacts DNA. Hence, Rec102 has a key DNA-binding function that is important for DSB formation.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 18/01/2026

In Topo VI, the B subunit contributes to DNA binding, so we asked whether the related subunit Rec102 shares this function. Lacking decent structural models at the time, we mutated several positive patches in Rec102, and identified one interesting candidate that kills DSB formation.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 18/01/2026

In parallel, a forward genetic screen identified a mutation within Spo11 that abolishes DSB formation. We showed that this mutation does not affect DNA binding and dimerization, but creates a steric clash across the dimer interface that obstructs the active site and abolishes cleavage.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 18/01/2026

We showed by gel shift that this mutation does not impact the affinity of the Spo11 core complex for DNA, but specifically reduces the assembly of dimeric complexes.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 18/01/2026

We mutated dozens of residues at the predicted dimer interface, and were surprised to find that most mutations had no meiotic phenotype. Nevertheless, we found one triple mutant that significantly reduces DSB formation and leads to reduced spore viability.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 18/01/2026

To gain further insights into Spo11 dimerization, we modeled the structure of a DNA-bound dimeric core complex. AlphaFold3 produced a high-confidence model, showing the DNA substrate bent at an angle of about 100°, consistent with previous AFM data.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 18/01/2026

We investigated how purified Spo11 core complex bind DNA in vitro using gel shift assays. We found that it indeed dimerizes on DNA to form an unstable complex that repeatedly dissociates and reassembles during electrophoresis.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 18/01/2026

We previously showed that the Spo11 core complex binds DNA and hypothesized that it may dimerize on DNA. Based on AFM imaging, we also proposed that the Spo11 complex bends DNA and perhaps traps a second DNA duplex prior to DNA cleavage, like type II topoisomerases do.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 18/01/2026

Spo11 cleaves DNA via a topoisomerase-like mechanism involving hybrid active sites located at the dimer interface. Spo11 forms a complex with Ski8, Rec102 and Rec104, but unlike its topoisomerase relative, the complex has a monomeric (1:1:1:1) stoichiometry. How does it dimerize?

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

We think that the binding of Spp1 to Mer2 occludes part of the DNA-binding interface of Mer2. However, the cost associated to Spp1 binding is compensated by a DNA-binding motif contributed by Spp1. We refer to this as an occlusion-compensation model.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

So how does Spp1 bind DNA? Again AlphaFold proved helpful and pointed to a motif required for DNA binding. Mutating this motif confirmed that, although full-length Spp1 does not bind DNA by itself, it binds DNA in the context of a complex with Mer2.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

While full-length Spp1 does not bind DNA by itself, a truncation of Spp1 bound DNA quite efficiently. Thus, DNA-binding appears to be auto-inhibited in the context of the full-length protein. That was surprise 3.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

Mer2 forms DNA-dependent condensates and effectively recruits Spp1. Spp1 is essentially recruited as a client, but Spp1 does seem to stimulate condensation a little.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

Now on two the second surprise: The Mer2 coiled coil domain that binds Spp1 is also involved in DNA binding. We reconstituted Mer2-Spp1-DNA complexes and used AlphaFold to visualize what these complexes might look like. (these models look pretty cool but are to be taken with a big grain of salt)

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

We verified that the previously-reported 4:2 stoichiometry was right. Indeed it was. Yet SAXS analysis all fit with the AlphaFold model. There are several possible explanations: Our favorite is that the binding of Spp1 to Mer2 is allosterically regulated. (our arguments are in the paper).

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

However, there is an issue: based on the AlphaFold model, it is not clear why Mer2 and Spp1 would assemble a complex with a 4:2 stoichiometry, as had been shown previously. Mer2 is a homotetramer, and based on the model, it could accommodate 4 Spp1 subunits. That's our 1st surprise.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

An mutagenesis analysis of the predicted interface provided evidence that supports the model.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

We used AlphaFold to model the structure of the Mer2-Spp1 interaction domain, revealing a cool model with two Spp1 bound to a tetrameric Mer2 coiled coil.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

We set out to characterize the interaction between Mer2 and Spp1. We developed an approach based on atomic-force microscopy to measure single-molecule Spp1-Mer2 interactions, revealing a dynamic interaction.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 28/09/2025

Meiotic double-strand break (DSB) formation is tied to the loop-axis organization of meiotic chromosomes. DSB proteins are localized to the axes, and Spo11 cleaves DNA within loops. Spp1 connects the loop to the axes by binding H3K4me3 marks within loops and the DSB protein Mer2 on the axis.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 19/02/2025

Adding to this model, we now propose that condensates recruit SPO11-TOP6BL complexes, thereby increasing local SPO11 concentration, allowing dimerization and cleavage.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 19/02/2025

SPO11 and its partner TOP6BL form a 1:1 complex of similar cleavage activity to SPO11 alone, although dependent on the experimental condition. SPO11-TOP6BL complexes bind DNA ends with high affinity, suggesting a possible role after cleavage, while SPO11 alone does not.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 19/02/2025

In vivo, SPO11 requires multiple other partners. These are dispensable in vitro because SPO11 monomers can dimerize at high protein concentration, probably through direct encounters on the DNA substrate.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 19/02/2025

In conditions that accumulate single-strand nicks, we detect a weak plasmid relaxation activity, likely caused by dissociation of the SPO11 dimer interface, swiveling around the intact strand, and re-ligation. Hence, cleavage is inherently reversible.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 19/02/2025

Purified SPO11 is mostly monomeric in vitro, but must dimerize to create two hybrid active sites, each responsible to cut one DNA strand.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 19/02/2025

We found that the selection of cleavage sites is affected by a combination of interrelated factors, including DNA sequence, topology and bendability.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 19/02/2025

And creates breaks with 2-nucleotides 5' overhangs.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 19/02/2025

As expected from in vivo data, we found that SPO11 remains covalently bound to the 5’ DNA ends.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 19/02/2025

We purified mouse SPO11 protein and found a robust plasmid DNA cleavage activity that depended on the active site tyrosine and metal ions.

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Corentin Claeys Bouuaert @ccb-lab.bsky.social · 23/12/2024

🥳 Paper accepted just on time for Christmas! Happy Holidays from the CCB lab! 🎄 🎅🏻 🥂 💫

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