BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Mutations in the breast cancer susceptibility gene, BRCA2, increase lifetime risk for breast and ovarian cancers.
• BRCA2 is crucial for DNA repair via homologous recombination, a process involving RAD51 nucleoprotein filament assembly on single-stranded DNA (ssDNA).
• Replication protein-A (RPA) binds ssDNA, creating a barrier to RAD51 filament formation that suppresses unregulated recombination.

Purpose of the Study:

• To directly measure the binding of BRCA2 and the assembly of RAD51 filaments on RPA-coated ssDNA.
• To elucidate the mechanism by which BRCA2 overcomes the RPA-imposed kinetic barrier to RAD51 filament formation.

Main Methods:

• Utilized a combination of microfluidics, microscopy, and micromanipulation techniques.
• Studied individual DNA molecules mimicking resected DNA lesions in replication-coupled recombinational repair.
• Measured BRCA2 binding and RAD51 filament assembly on RPA-coated ssDNA.

Main Results:

• A dimer of RAD51 is minimally required for spontaneous nucleation, but filament growth self-terminates.
• BRCA2 accelerates RAD51 nucleation to overcome the RPA kinetic block, approaching the rate of RAD51 association with naked ssDNA.
• BRCA2 chaperones a preassembled RAD51 filament onto RPA-complexed ssDNA, bypassing the rate-limiting nucleation step.

Conclusions:

• BRCA2 acts as a recombination mediator by initiating RAD51 filament formation on RPA-coated ssDNA.
• BRCA2's mechanism involves accelerating nucleation and chaperoning preassembled filaments, thereby regulating homologous recombination.
• Understanding BRCA2's function is critical for comprehending DNA repair and its role in preventing cancer.