Reconstitution of SPO11-dependent double-strand break formation

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Homologous meiotic recombination initiates with DNA double-strand breaks (DSBs) created by the SPO11 protein.
• SPO11 is vital for meiosis but its DSBs pose risks due to mutagenic and gametocidal potential, necessitating strict cellular control.
• The precise mechanism and regulation of SPO11 activity remain incompletely understood.

Purpose of the Study:

• To reconstitute and characterize the DNA cleavage activity of mouse SPO11 in vitro.
• To elucidate the structural and mechanistic requirements for SPO11-mediated DNA double-strand break formation.
• To understand the regulation of SPO11 activity and its dependence on dimerization and accessory proteins.

Main Methods:

• Purification and in vitro reconstitution of mouse SPO11-TOP6BL complexes.
• DNA cleavage assays using purified recombinant proteins.
• Structure modeling using AlphaFold3.
• Deep sequencing of in vitro cleavage products.

Main Results:

• Mouse SPO11-TOP6BL complexes form dimers (2:2) that cleave DNA, requiring SPO11 active site residues, metal ions, and dimerization.
• SPO11 exhibits both DNA cleavage and topoisomerase activity (relaxing supercoils and resealing nicks).
• DNA bending precedes cleavage, and cleavage site preference is influenced by base composition bias.
• SPO11 activity is inefficient on complex substrates due to slow exchange from monomeric states, but can be enhanced by promoting dimer formation.

Conclusions:

• SPO11's intrinsic dimerization is weak, acting as a restraint on its activity in vivo.
• Accessory proteins are crucial for focusing and controlling SPO11-mediated DSB formation at specific times and locations during meiosis.
• This study provides mechanistic insights into DSB formation and regulation, crucial for maintaining genomic stability during reproduction.