RAD51C-XRCC3 structure and cancer patient mutations define DNA replication roles

Area of Science:

• Molecular Biology
• Structural Biology
• Cancer Genetics

Background:

• RAD51C is a gene linked to hereditary breast, ovarian, and prostate cancers.
• Current understanding of RAD51C mutations is limited to its role in homology-directed repair (HDR).
• A lack of structural and functional data hinders the interpretation of patient-derived RAD51C mutations.

Purpose of the Study:

• To elucidate the three-dimensional structure of the RAD51C-XRCC3 (CX3) complex.
• To define the distinct roles of RAD51C in DNA replication stability.
• To correlate structural findings with cancer patient mutation data for functional interpretation.

Main Methods:

• X-ray co-crystallography of the RAD51C-XRCC3 (CX3) complex with an ATP analog.
• CRISPR/Cas9 gene editing in human cells to create RAD51C mutations.
• Single-molecule, single-cell, and biophysical measurements to assess protein function.

Main Results:

• The study determined the X-ray crystal structure of the RAD51C-XRCC3 (CX3) complex.
• Distinct functional regions within CX3 were identified, mediating DNA replication fork protection, restart, and reversal.
• Cancer patient mutations were mapped onto the structure, revealing specific interfaces related to ATP-binding and DNA interactions.

Conclusions:

• The RAD51C-XRCC3 (CX3) complex functions as a replication stress response unit.
• Separable roles in DNA binding and filament capping contribute to replication stability.
• Understanding these distinct functions is crucial for interpreting RAD51C mutations in cancer predisposition and development.