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bioRxiv · 10.1101/2024.11.20.624382

Reconstitution of SPO11-dependent double-strand break formation

Zheng, Z.·Zheng, L.·Arter, M.·Liu, K.·Yamada, S.·Ontoso, D.·Kim, S.·Keeney, S.
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Abstract

Homologous meiotic recombination starts with DNA double-strand breaks (DSBs) generated by SPO11 protein1. SPO11 is critical for meiosis in most species but the DSBs it makes are also dangerous because of their mutagenic2 and gametocidal3 potential, so cells must foster SPO11s beneficial functions while minimizing its risks4. SPO11 mechanism and regulation remain poorly understood. Here we report reconstitution of DNA cleavage in vitro with purified recombinant mouse SPO11 bound to its essential partner TOP6BL. Similar to their yeast orthologs5,6, SPO11- TOP6BL complexes are monomeric (1:1) in solution and bind tightly to DNA. Unlike in yeast, however, dimeric (2:2) assemblies of mouse SPO11-TOP6BL cleave DNA to form covalent 5' attachments requiring SPO11 active site residues, divalent metal ions, and SPO11 dimerization. Surprisingly, SPO11 can also manifest topoisomerase activity by relaxing supercoils and resealing DNA that it has nicked. Structure modeling with AlphaFold37 illuminates the protein-DNA interface and suggests that DNA is bent prior to cleavage. Deep sequencing of in vitro cleavage products reveals a rotationally symmetric base composition bias that partially explains DSB site preferences in vivo. Cleavage is inefficient on complex DNA substrates, partly because SPO11 is readily trapped in DSB-incompetent (presumably monomeric) binding states that exchange slowly. However, cleavage is improved by using substrates that favor DSB-competent dimer assembly, or by fusing SPO11 to an artificial dimerization module. Our results inform a model in which intrinsically feeble dimerization restrains SPO11 activity in vivo, making it exquisitely dependent on accessory proteins that focus and control DSB formation so that it happens only at the right time and the right places.

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Zheng, Z., Zheng, L., Arter, M., Liu, K., Yamada, S., Ontoso, D., Kim, S., Keeney, S.. 2024-11-21. Reconstitution of SPO11-dependent double-strand break formation. https://doi.org/10.1101/2024.11.20.624382

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