RAD51AP1 is a versatile RAD51 modulator

RAD51AP1 is an emergent key factor in homologous recombination (HR), the major pathway for accurate repair of DNA double-strand breaks, and in alternative lengthening of telomeres (ALT). Depletion of RAD51AP1 diminishes HR and overexpression is common in cancer, where it is associated with malignancy. Here, we show that RAD51AP1 serves as a versatile modulator of the RAD51 recombinase, the central player in HR. Through a combination of biochemistry and structural biology, we reveal that RAD51AP1 possesses at least three RAD51-binding sites that facilitate its binding across two adjacent RAD51 molecules. We uncover a novel RAD51 binding mode and characterise a hitherto overlooked role for RAD51AP1 in stabilising RAD51-ssDNA filaments and promoting strand exchange. Further, we resolved structures of RAD51-ssDNA filaments in the presence of Mg ²⁺ -ATP and Mg ²⁺ -ADP, revealing conformational changes upon ATP hydrolysis and explaining how ADP reduces RAD51-DNA binding. Our findings provide mechanistic insights into RAD51 recombinase and RAD51AP1.