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 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. The in vitro characterization of a key event in the early stages of meiosis—the induction of double-strand DNA breaks by the SPO11–TOP6BL complex—provides insight into the catalytic mechanism and evolution of SPO11.