Researchers reconstitute meiotic double-strand break formation in vitro

A double-strand break (DSB) is a type of DNA damage where both strands of the DNA helix are cut or broken at the same location, causing a complete discontinuity in the DNA molecule. Unrepaired or improperly repaired DSBs can lead to mutations, chromosomal rearrangements, or cell death. They may be associated with cancer, aging, and genetic disorders. However, they are also essential for meiosis—the process of forming sperm and egg cells.

In the latter situation, DSBs are intentionally introduced to allow genetic recombination by mixing parental genes. Homologous recombination—a crucial meiotic event—not only enhances genetic diversity through exchanging parental genetic material but also establishes physical connections between homologous chromosomes to ensure their precise segregation. 

As long ago as 1997, researchers understood that generation of programmed DSBs required by homologous recombination was catalyzed by SPO11. Nevertheless, despite nearly three decades of effort, researchers were unable to reconstitute in vitro the formation of DSB until a recent breakthrough by Chinese, American and Belgian scientists.

In a study published in Nature on February 19, researchers led by Prof. TONG Minghan from the Center for Excellence in Molecular Cell Science (Shanghai Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences, collaborating with Prof. HUANG Ying from Xinhua Hospital, Shanghai Jiaotong University School of Medicine, reported the successful in vitro reconstitute of the meiotic DNA double-strand break (DSB) formation.

In this study, researchers expressed and purified the SPO11-TOP6BL complex using Expi293F cells. They demonstrated that this complex cleaves DNA and covalently attaches to the 5' terminus of DNA breaks in vitro, thus successfully reconstitute the meiotic DSB formation in vitro.

Using a point-mutation strategy, the researchers revealed that Mg²⁺ is essential for the complex's DNA cleavage activity. Knock-in mice carrying a SPO11 point-mutation that disrupts Mg²⁺ binding exhibited a complete loss of DSB formation. Interestingly, the activity of the SPO11 complex functions independently of ATP, in contrast with its ancestral enzyme, topoisomerase VI.

This study represents a major breakthrough in understanding meiotic recombination. It provides a powerful platform for dissecting the molecular mechanisms of meiotic homologous recombination.