Structures and molecular mechanisms of RAD54B in modulating homologous recombination

Genome stability is essential for cellular viability yet constantly threatened by DNA-damaging agents. Among these, DNA double-strand breaks (DSBs) are particularly harmful and in S/G2 phases are faithfully repaired through homologous recombination (HR). The RAD51 recombinase forms nucleoprotein filaments on single-stranded DNA (ssDNA) that performs critical functions in HR. The efficiency of HR depends on precise regulation of RAD51 filament dynamics by accessory factors, including RAD54B, a double-strand DNA-dependent ATPase. Here, using cryogenic electron microscopy, mutagenesis, and biochemical assays, we show that RAD54B stabilizes RAD51–DNA filaments, inhibits RAD51 ATPase activity, and promotes D-loop formation and strand exchange. RAD54B contains two main RAD51-interacting sites and a β-domain that bridges RAD51 protomers, is positioned to contact donor dsDNA, and regulates RAD54B’s ATPase activity. Cellular assays reveal that the RAD51-interacting sites are required for repairing DSBs by HR in human cells. Our findings uncover a modular architecture and mechanistic framework for RAD54B’s critical role in genome maintenance.