ssDNA hand-off from RPA to Polθ drives microhomology-mediated end-joining

DNA polymerase theta-mediated end joining (TMEJ) is the primary mechanism of double strand break repair in mitosis and becomes essential in BRCA-deficient cells. RPA, the ssDNA-binding protein that coats resected DNA ends, has long been thought to inhibit this pathway by blocking the initial microhomology search in TMEJ. Here we find instead that RPA plays an essential activating role in TMEJ. Structural, biochemical, and genetic analyses support a model in which Polθ binds directly to RPA at resected DNA ends and then competes with RPA for ssDNA. This releases ssDNA into the Polθ helicase domain. We propose that threading two DNA ends through the Polθ dimer in this manner initiates the search for microhomology. Together, our findings establish a detailed mechanism of how proteins can harness RPA to access ssDNA for critical genome maintenance transactions.