Riding the Fork: Defining the replication-coupled resection complex that bypasses canonical BRCA1 control

Pavani et al. (Science, 2024) showed that fork collapse at leading- vs lagging-strand nicks yields distinct DSB end structures and that end resection at these replication-coupled breaks is BRCA1-independent, with BRCA1 acting downstream to counter 53BP1’s suppression of RAD51 loading. This project proposes to biochemically capture and reconstitute the nucleases/cofactors that travel with the fork and initiate resection upon collapse. It will combine synchronized nCas9 nicking with APEX2/TurboID fused to PCNA or replisome components for fork-proximal proteomics, plus single-molecule imaging of RAD51 filament dynamics. Repair outcomes will be quantified by long-read sequencing and microhomology usage, benchmarking against MMEJ/SSA biases. This approach translates the conceptual advance of BRCA1-independent resection at fork-coupled breaks into a molecular parts list and kinetic model, linking outcomes to mutational signatures distinct from standard IR/endonuclease-induced breaks. A fork-specific resection complex could be druggable in HR-deficient tumors or normal tissues, explaining distinctive microhomology-rich scars in HR-deficient cancers. The impact includes a refined mechanistic model of replication-coupled DSB processing that can improve genome editing strategies and predict tumor evolution under PARP/TOP1 inhibitors.

References:

1. Structure and repair of replication-coupled DNA breaks. R. Pavani, Veenu Tripathi, Kyle B Vrtis, D. Zong, R. Chari, E. Callen, Ajith V. Pankajam, Gang Zhen, Gabriel E. Matos-Rodrigues, Jiajie Yang, Shuheng Wu, Giordano Reginato, Wei Wu, Petr Cejka, Johannes C. Walter, A. Nussenzweig (2024). Science.