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

The Spo11 complex catalyses the formation of DNA double-strand breaks (DSBs), initiating meiotic recombination-a process that is essential for fertility and genetic diversity^1,2. 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.