A direct RPA-Polθ interaction licenses theta-mediated end joining

DNA double-strand break repair often involves end resection, yet how resected ends are directed toward distinct repair pathways remains poorly understood. Here, we identify replication protein A (RPA) as an unexpected essential factor for cellular polymerase theta-mediated end joining (TMEJ). Using MUSIC-MAP, a newly developed platform linking endogenous sequence variants to DNA repair outcomes, we map a discrete functional interface between RPA2 and polymerase theta (Pol{theta}). Disrupting this interface in RPA2 abolishes TMEJ without impairing cell viability or homologous recombination. Mutations in the complementary Pol{theta} surface likewise impair TMEJ, whereas reciprocal charge-reversal mutations in RPA2 and Pol{theta} restore TMEJ, providing genetic evidence for a direct physical interaction. Structural modeling suggests that the Pol{theta}-binding surface becomes accessible at RPA-coated ssDNA termini, positioning RPA as a docking platform for Pol{theta} engagement. These findings reveal how RPA, previously considered a barrier to TMEJ, instead functions as a molecular platform that translates the organization of ssDNA into selective enzyme engagement, enabling mutagenic repair without compromising homologous recombination.