Human RAD51 Protein Forms Amyloid-like Aggregates In Vitro

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RAD51 is crucial for homologous recombination and DNA double-strand break (DSB) repair, maintaining genome stability.
• Amyloids are protein aggregates with specific structural and functional properties, often associated with diseases but also with biological roles.

Purpose of the Study:

• To investigate the in vitro amyloid properties of the human RAD51 protein.
• To assess RAD51's aggregation behavior in a bacterial curli-dependent amyloid generator (C-DAG) system.
• To explore the potential biological relevance of RAD51's amyloid characteristics.

Main Methods:

• In vitro biochemical assays including detergent resistance, amyloid-specific dye staining (e.g., Congo red), and polarized microscopy.
• Structural analyses using transmission electron microscopy (TEM) and X-ray diffraction.
• In vivo assessment in a bacterial C-DAG system and observation of RAD51 aggregates in overexpressing cell cultures.

Main Results:

• Purified human RAD51 formed detergent-resistant aggregates in vitro with an unbranched cross-β fibrillar structure, characteristic of amyloids.
• RAD51 aggregates stained positively with amyloid-specific dyes and exhibited birefringence under polarized light.
• X-ray diffraction confirmed a cross-β structure in RAD51 fibrils, and cytoplasmic aggregates were observed in vivo.

Conclusions:

• The study demonstrates that RAD51 possesses amyloid properties in vitro and in a bacterial system.
• These findings reveal a novel aspect of RAD51's molecular behavior, suggesting potential implications for its role in genome stability and other cellular processes.