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

• 成果类型: Article
• 署名作者: Tang, Xinzhe; Hu, Zetao; Ding, Jian; Wu, Meixia; Guan, Pin; Song, Yawei; Yin, Yue; Wu, Wei; Ma, Jinbiao; Huang, Ying; Tong, Ming-Han
• 署名单位: Chinese Academy of Sciences; Center for Excellence in Molecular Cell Science, CAS; University of Chinese Academy of Sciences, CAS; Shanghai Jiao Tong University; Fudan University; Chinese Academy of Sciences; Shanghai Advanced Research Institute, CAS
• 刊物名称: Nature
• ISSN/ISSBN: 0028-2091
• DOI: 10.1038/s41586-024-08551-1
• 发表日期: 2025-03-20
• 关键词: topoisomerase-ii; spo11 gene; recombination; prdm9

摘要

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.