In vitro reconstitution of meiotic DNA double-strand-break formation

Xinzhe Tang, Zetao Hu, Jian Ding, Meixia Wu, Pin Guan, Yawei Song, Yue Yin, Wei Wu, Jinbiao Ma, Ying Huang () and Ming-Han Tong ()
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Xinzhe Tang: University of Chinese Academy of Sciences
Zetao Hu: Shanghai Jiao Tong University School of Medicine
Jian Ding: University of Chinese Academy of Sciences
Meixia Wu: University of Chinese Academy of Sciences
Pin Guan: University of Chinese Academy of Sciences
Yawei Song: University of Chinese Academy of Sciences
Yue Yin: Chinese Academy of Sciences
Wei Wu: University of Chinese Academy of Sciences
Jinbiao Ma: Fudan University
Ying Huang: Shanghai Jiao Tong University School of Medicine
Ming-Han Tong: University of Chinese Academy of Sciences

Nature, 2025, vol. 639, issue 8055, 800-807

Abstract: Abstract The Spo11 complex catalyses the formation of DNA double-strand breaks (DSBs), initiating meiotic recombination—a process that is essential for fertility and genetic diversity1,2. Although the function of Spo11 has been known for 27 years, previous efforts to reconstitute DSB formation in vitro have been unsuccessful. Here we biochemically characterize the mouse SPO11–TOP6BL protein complex, and show that this complex cleaves DNA and covalently attaches to the 5′ terminus of DNA breaks in vitro. Using a point-mutation strategy, we reveal that Mg2+ is essential for the DNA-cleavage activity of this complex in vitro, as confirmed by knock-in mice carrying a point mutation in SPO11 that disrupts its binding to Mg2+, thereby abolishing DSB formation. However, the activity of the SPO11 complex is ATP-independent. We also present evidence that the mouse SPO11 complex is biochemically distinct from the ancestral topoisomerase VI. Our findings establish a mechanistic framework for understanding the first steps of meiotic recombination.

Date: 2025
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DOI: 10.1038/s41586-024-08551-1

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