In vitro reconstitution of meiotic DNA double-strand break formation

The Spo11 complex catalyzes the formation of DNA double-strand breaks (DSBs), initiating meiotic recombination-a process essential for fertility and genetic diversity. Although Spo11's function has been known for 27 years, previous efforts to reconstitute DSB formation in vitro have been unsuccessful. Here, we biochemically characterize mouse SPO11 and TOP6BL protein complex and demonstrate 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 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 initial steps of meiotic recombination. Overall design: We employed END-seq to map DNA cleavage sites with high resolution, aiming to elucidate whether the SPO11-TOP6BL complex exhibits DNA sequence specificity in its activity.