The chapter discusses the mechanism of single-stranded DNA (ssDNA) annealing by the RAD52–RPA complex. RAD52 plays a crucial role in DNA repair, mitotic DNA synthesis, and alternative telomere length maintenance. It promotes ssDNA annealing and is an attractive therapeutic target for homologous recombination-deficient cancers. The structure of RAD52 reveals that it forms undecameric rings, but the active form is an open ring associated with replication protein A (RPA). Cryo-electron microscopy and biochemical analyses show that ssDNA binds to the positively charged channel around the RAD52 ring, driven by the N-terminal domains. The C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the ring opening site, with critical interactions between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. The RAD52–RPA complex facilitates ssDNA annealing by bringing complementary sequences into close proximity, leading to the formation of duplex DNA. This mechanism suggests a new therapeutic strategy to inhibit RAD52–RPA interactions in homologous recombination-deficient cancer cells.The chapter discusses the mechanism of single-stranded DNA (ssDNA) annealing by the RAD52–RPA complex. RAD52 plays a crucial role in DNA repair, mitotic DNA synthesis, and alternative telomere length maintenance. It promotes ssDNA annealing and is an attractive therapeutic target for homologous recombination-deficient cancers. The structure of RAD52 reveals that it forms undecameric rings, but the active form is an open ring associated with replication protein A (RPA). Cryo-electron microscopy and biochemical analyses show that ssDNA binds to the positively charged channel around the RAD52 ring, driven by the N-terminal domains. The C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the ring opening site, with critical interactions between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. The RAD52–RPA complex facilitates ssDNA annealing by bringing complementary sequences into close proximity, leading to the formation of duplex DNA. This mechanism suggests a new therapeutic strategy to inhibit RAD52–RPA interactions in homologous recombination-deficient cancer cells.