The human RAD52 complex undergoes phase separation and facilitates bundling and end-to-end tethering of RAD51...

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• Human RAD52 is a target for synthetic lethality in homologous recombination-deficient cancers.
• RAD52's functions in DNA repair and replication fork protection can substitute for BRCA2.
• Mechanisms of RAD52's BRCA2-like functions are not fully understood.

Purpose of the Study:

• Investigate the macromolecular organization and phase separation capabilities of human RAD52.
• Elucidate how RAD52's structure and interactions contribute to homologous recombination.
• Determine RAD52's role in organizing RAD51 nucleoprotein filaments.

Main Methods:

• Homotypic phase separation assays.
• Fluorescence microscopy.
• Single-molecule super-resolution imaging (DNA-PAINT).
• Atomic force microscopy.

Main Results:

• RAD52 exhibits homotypic phase separation, forming condensates that recruit ssDNA, RPA, and RAD51.
• RAD52 phase separation is regulated by ssDNA and RPA.
• RAD52 induces the formation of RAD51-ssDNA fibrillar structures, which are bundles of RAD51 filaments.
• RAD52 mediates end-to-end tethering of RAD51 nucleoprotein filaments.

Conclusions:

• RAD52 possesses unique self-assembly properties through phase separation.
• These properties enable RAD52 to organize key homologous recombination proteins, including RAD51 filaments.
• RAD52's macromolecular organization is critical for its functions in DNA repair and potentially its role in compensating for BRCA2 loss in cancer.