Corentin Claeys Bouuaert

Guest post: Forget pickles and ice cream. I published a fake paper on pregnancy cravings for prime numbers Image generated by Google Gemini I had grown weary of the constant stream and abuse of spam invitations to submit manuscripts to journals and to attend fake conferences on the other side of the wor…

Forget pickles and ice cream. I published a fake paper on pregnancy cravings for prime numbers.

This work was led by Hajar Aït Bella, with key contributions from lab members Mahesh Survi and Julian Urdiain-Arraiza, and from the Hochwagen lab. Congratulations to all, and thanks to the ERC @erc.europa.eu and the FNRS for funding!

To conclude, our structure-function analysis provides new insights into how Spo11 dimerizes and how its partners contribute to the initiation of meiotic recombination.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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?

I’m happy to present a new paper from the lab, where we investigated how the yeast Spo11 core complex dimerizes to induce the formation of meiotic DNA double-strand breaks. 🧵

www.biorxiv.org/content/10.6...

IT'S HAPPENING! 💥 I'm psyched to launch the collaboration between @qedscience.bsky.social & @openrxiv.bsky.social @biorxivpreprint.bsky.social! Preprint + q.e.d = your science is out there, and anyone can appreciate it. Let's care about making discoveries, and not on “getting published” (1/3) 👇

Nature suggests you use their "Manuscript Adviser" bot to get advice before submitting

I uploaded the classic Watson & Crick paper about DNA structure, and the Adviser had this to say about one of the greatest paper endings of the century:

Among the anti-recombinases, FIGNL1 rules them all. So much that inactivating it brings BRCA2-deficient cells to life. Who is responsible for RAD51 loading without BRCA2/FIGNL1, check out the paper to find out! Great collaboration with @raychaudhurilab.bsky.social

www.science.org/doi/10.1126/...

This work was led by my research technician Pascaline Liloku. Congratulations to her! I thank our collaborators, in particular the team of David Alsteens for help with AFM experiments, and Yann Sterckx for SAXS experiments. Finally, big thanks to the ERC and FNRS for funding!

Do we have any evidence to support this?

And does the DNA-binding activity of Spp1 have any functional consequences?

To find out, check out the paper!

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.

So, how then do Mer2-DNA and Mer2-Spp1 interactions relate to each other? In other words, are the binding of DNA and Spp1 to Mer2 independent? Or are they competitive? Or in contrast are they cooperative?

We think there is yet another possibility.

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.

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.

So how does Spp1 affect DNA binding and condensation?

We expected that DNA and Spp1 might compete for access to Mer2, but that doesn't seem to be the case. Instead Spp1 seems to somewhat stimulate DNA binding and condensation by Mer2.

Does Spp1 contribute to DNA binding directly?

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.

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)

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).

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.

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

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.