Reconstitution of SPO11-dependent double-strand break formation

Zhi Zheng, Lyuqin Zheng, Meret Arter, Kaixian Liu, Shintaro Yamada, David Ontoso, Soonjoung Kim and Scott Keeney ()
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Zhi Zheng: Memorial Sloan Kettering Cancer Center
Lyuqin Zheng: Memorial Sloan Kettering Cancer Center
Meret Arter: Memorial Sloan Kettering Cancer Center
Kaixian Liu: Memorial Sloan Kettering Cancer Center
Shintaro Yamada: Memorial Sloan Kettering Cancer Center
David Ontoso: Memorial Sloan Kettering Cancer Center
Soonjoung Kim: Memorial Sloan Kettering Cancer Center
Scott Keeney: Memorial Sloan Kettering Cancer Center

Nature, 2025, vol. 639, issue 8055, 784-791

Abstract: Abstract Meiotic recombination starts with SPO11 generation of DNA double-strand breaks (DSBs)1. SPO11 is critical for meiosis in most species, but it generates dangerous DSBs with mutagenic2 and gametocidal3 potential. Cells must therefore utilize the beneficial functions of SPO11 while minimizing its risks4—how they do so remains poorly understood. Here we report reconstitution of DNA cleavage in vitro with purified recombinant mouse SPO11 bound to TOP6BL. SPO11–TOP6BL complexes are monomeric (1:1) in solution and bind tightly to DNA, but dimeric (2:2) assemblies cleave DNA to form covalent 5′ attachments that require SPO11 active-site residues, divalent metal ions and SPO11 dimerization. SPO11 can also reseal DNA that it has nicked. Structure modelling with AlphaFold 3 suggests that DNA is bent prior to cleavage5. In vitro cleavage displays a sequence 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 with substrates that favour dimer assembly or by artificially dimerizing SPO11. Our results inform a model in which intrinsically weak dimerization restrains SPO11 activity in vivo, making it exquisitely dependent on accessory proteins that focus and control DSB formation.

Date: 2025
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DOI: 10.1038/s41586-025-08601-2

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