The 9-1-1 complex protects ssDNA gaps in BRCA2-deficient cancer

Single-stranded DNA (ssDNA) gaps are a hallmark of BRCA-deficient cells, yet the mechanisms that safeguard these lesions remain unclear. Using a genome-wide CRISPR screen in isogenic BRCA2-proficient and -deficient cancer cells, we identified the RAD9A-HUS1-RAD1 (9-1-1) complex as essential for BRCA2-deficient cell survival through an ATR-independent mechanism. In this context, loss of 9-1-1 leads to accumulation of PRIMPOL-dependent gaps that fail to undergo post-replicative repair, resulting in pathological expansion and increased DNA damage. Excessive EXO1-mediated degradation drives this genomic instability, as EXO1 depletion rescues the phenotype. We further demonstrate that the 9-1-1 complex contributes to POLζ-dependent gap filling. We propose a model in which ssDNA gaps, when extended beyond a critical length, become inaccessible to translesion synthesis (TLS)-mediated repair and rely entirely on homologous recombination. These findings establish the 9-1-1 complex as a key regulator of ssDNA gap stability and as a promising therapeutic target in BRCA2-deficient cancers.