In Vitro Reconstitution of Meiotic DNA Double-strand-break Formation

Overview

Journal Nature

Specialty Science

Date 2025 Feb 19

PMID 39972125

Authors

Xinzhe Tang

Zetao Hu

Jian Ding

Meixia Wu

Pin Guan

Yawei Song

Yue Yin

Wei Wu

Jinbiao Ma

Ying Huang

Ming-Han Tong

Affiliations

Soon will be listed here.

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 diversity. 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 Mg 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 Mg, 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.

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