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 (Polθ). Disrupting this interface in RPA2 abolishes TMEJ without impairing cell viability or homologous recombination. Mutations in the complementary Polθ surface likewise impair TMEJ, whereas reciprocal charge-reversal mutations in RPA2 and Polθ restore TMEJ, providing genetic evidence for a direct physical interaction. Structural modeling suggests that the Polθ-binding surface becomes accessible at RPA-coated ssDNA termini, positioning RPA as a docking platform for Polθ 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.