Reconstitution of SPO11-dependent double-strand break formation

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Meiotic recombination initiates with SPO11-generated DNA double-strand breaks (DSBs).
• SPO11 is essential for meiosis but poses risks due to DSBs' mutagenic and gametocidal potential.
• Mechanisms controlling SPO11's dual functions remain unclear.

Purpose of the Study:

• To reconstitute and characterize DNA cleavage activity of mouse SPO11 in vitro.
• To elucidate the role of SPO11 dimerization and TOP6BL in regulating DSB formation.
• To understand the sequence specificity and substrate preferences of SPO11.

Main Methods:

• Purification and in vitro reconstitution of mouse SPO11-TOP6BL complexes.
• DNA cleavage and resealing assays.
• AlphaFold 3 structure modeling.
• Analysis of sequence bias and substrate efficiency.

Main Results:

• SPO11-TOP6BL complexes bind DNA, with dimeric assemblies (2:2) cleaving DNA and forming covalent 5' attachments.
• Cleavage requires SPO11 active-site residues, metal ions, and dimerization; SPO11 can also reseal nicks.
• In vitro cleavage shows sequence bias and is inefficient on complex substrates due to slow monomer-dimer exchange, but improved by promoting dimerization.

Conclusions:

• Weak intrinsic dimerization restrains SPO11 activity, necessitating accessory proteins for focused and controlled DSB formation in vivo.
• This provides insight into how cells manage SPO11's essential but hazardous role in meiosis.
• Understanding SPO11 regulation is key to meiosis and preventing associated genetic risks.