Michael A. Longo¹ ∙ Syed Moiz Ahmed^2,10 ∙ Yue Chen^3,10 ∙ Chi-Lin Tsai^1,10 ∙ Sarita Namjoshi¹ ∙ Runze Shen¹ ∙ Zamal Ahmed¹ ∙ Xiaoyan Wang¹ ∙ Rajika L. Perera⁴ ∙ Andy Arvai⁵ ∙ Miyoung Lee⁶ ∙ Li Ren Kong^2,6 ∙ Wilfried Eng^l7 ∙ Woei Shyuan Ng⁷ ∙ Ziqing Winston Zhao^7,8 ∙ Ashok R. Venkitaraman^2,6,9∙ John A. Tainer^1,3 ∙ Katharina Schlacher^3,11

1. Department of Molecular & Cellular Oncology, UT MD Anderson Cancer Center, Houston, TX 77030, USA
2. The Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599, Singapore
3. Department of Cancer Biology, UT MD Anderson Cancer Center, Houston, TX 77054, USA
4. Poseidon Laboratory, Pasadena, CA 91107, USA
5. The Department of Integrative Structural & Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
6. Medical Research Council Cancer Unit, University of Cambridge, Hills Road, Cambridge CB2 0XZ, UK
7. Department of Chemistry and Centre for BioImaging Sciences, National University of Singapore, Singapore 117543, Singapore
8. Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore
9. Institute of Molecular & Cell Biology, Agency for Science, Technology and Research (A∗STAR), Singapore 138673, Singapore
10. These authors contributed equally
11. Lead contact

Tumor suppressor protein breast cancer susceptibility protein 2 (BRCA2) acts with RAD51 in replication fork protection (FP) and homology-directed DNA-break repair (HDR). Critical for cancer etiology and therapy resistance, the BRCA2 C terminus was thought to stabilize recombinogenic RAD51 after the assembly of ATP-extended RAD51 filaments on single-stranded DNA (ssDNA). Here, the detailed crystal structure of the human BRCA2 C-terminal interaction domain (TR2 interface [TR2i]) complexed with ATP-bound RAD51 prior to DNA binding instead reveals TR2i unexpectedly induces a unique ATP-RAD51 dimer conformation that accommodates nucleation onto double-stranded B-DNA unsuited for HDR initiation. Structural, biochemical, and molecular results with interface-guided mutations uncover TR2i’s FP mechanism. Proline-driven secondary structure stabilizes residue triads and spans the RAD51 dimer, engaging pivotal interactions of RAD51 M210 and BRCA2 S3291/P3292, the cyclin-dependent kinase (CDK) phosphorylation site that toggles between FP during S phase and HDR in G2. TR2i evidently acts as an allosteric clamp, switching RAD51 from ssDNA to double-stranded and B-DNA binding, enforcing FP over HDR, challenging the current BRCA2-RAD51 dogma. DOI: 10.1016/j.molcel.2025.05.010