The repair of DNA double-strand breaks by homologous recombination is essential for genomic integrity, and its dysregulation is a hallmark of cancer1. Central to homologous recombination is the RAD51 recombinase, whose assembly into a nucleoprotein filament is governed by five RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3)2. Mutations in any of these proteins predispose individuals to multiple cancers or genetic disorders3–6. These paralogues are thought to form two functionally separate complexes RAD51B–RAD51C–RAD51D–XRCC2 (BCDX2) and RAD51C–XRCC3 (CX3), that act independently at different stages of homologous recombination7–11. Here we demonstrate that all five paralogues can assemble into a single, ATP-dependent BCDX2–CX3–RAD51 supercomplex. The architecture of this assembly bound to single-stranded DNA reveals a contiguous filament where the CX3 module stacks atop BCDX2, creating a protofilament template for RAD51 filament formation. We further identify a novel, RAD51B-independent DX2–CX3 complex (RAD51D–XRCC2–RAD51C–XRCC3) functioning as a stable RAD51 anchor on single-stranded DNA, and we capture it in multiple states, including capping RAD51 filament segment. These distinct assemblies are differentially regulated by ATPase activity, defining a dynamic BCDX2–CX3 ‘loader’ and a stable DX2–CX3 ‘anchor’ that provide functional modularity to the homologous recombination machinery. This work provides a unifying mechanism for human RAD51 paralogue function and delivers an atomic blueprint for interpreting disease-causing mutations.
