PARP inhibitor synthetic lethality reveals homologous recombination sub-pathway architecture

The DNA damage response (DDR) is a complex network of interconnected pathways and sub-pathways that safeguards genome integrity. Deciphering the coordinated and complementary interactions among these pathways remains a major challenge. In this study, we employed CRISPR screening to systematically map the genetic interactions required for different sub-pathways of homologous recombination in human cells following PARP inhibitor treatment. Our approach recapitulated known interactions and uncovered several previously unrecognized connections. We identified RAD54L, in addition to ATRX, as a factor promoting the double Holliday junction (dHJ) pathway and demonstrated that RAD51AP1 and RAD54B function in synthesis-dependent strand annealing (SDSA). We provide evidence that loss of TOP3A induces a switch in HR sub-pathway usage from SDSA to the dHJ pathway. Furthermore, TOP3A deficiency abolishes the requirement for ATRX and the histone variant H3.3 in the dHJ pathway, while maintaining strict dependence on RAD54L. We further observed that H3.3 is involved in both HR sub-pathways, whereas its depositing chromatin remodelers HIRA and ATRX play pathway-specific roles in SDSA and dHJ, respectively. Together, our findings define the architecture underlying HR sub-pathway choice and reveal a key role for TOP3A in regulating pathway balance.