BRCA2 C-terminal clamp restructures RAD51 dimers to bind B-DNA for replication fork stability

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• BRCA2 is a tumor suppressor crucial for DNA repair and cancer development.
• It functions with RAD51 in replication-fork protection (FP) and homology-directed DNA repair (HDR).
• The BRCA2 C-terminus was believed to stabilize RAD51 filaments on single-stranded DNA.

Purpose of the Study:

• To determine the crystal structure of the BRCA2 C-terminal interaction domain (TR2i) bound to RAD51 before DNA binding.
• To elucidate the mechanism by which BRCA2 regulates RAD51 activity in DNA repair pathways.

Main Methods:

• X-ray crystallography to obtain high-resolution structures.
• Biochemical assays to assess protein interactions and functions.
• Molecular analysis using interface-guided mutations.

Main Results:

• The crystal structure revealed that TR2i reshapes ATP-RAD51 into a unique dimer conformation.
• This conformation favors double-stranded B-DNA binding, which is unsuitable for HDR initiation.
• TR2i acts as an allosteric clamp, utilizing proline-driven structures and specific residue interactions (e.g., BRCA2 S3291/P3292) to switch RAD51.
• This switch enforces replication-fork protection (FP) during S-phase and HDR during G2, regulated by CDK phosphorylation.

Conclusions:

• BRCA2 TR2i allosterically regulates RAD51, switching its DNA binding preference from single-stranded to double-stranded DNA.
• This mechanism ensures replication-fork protection during S-phase and allows homology-directed repair in G2.
• The findings provide new insights into BRCA2's role in cancer etiology and therapy resistance.