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BCDX2-CX3 and DX2-CX3 complexes template RAD51 filament formation

ABSTRACT: The faithful repair of DNA double-strand breaks by homologous recombination (HR) is essential for genomic integrity, and its dysregulation is a hallmark of cancer. Central to HR is the RAD51 recombinase, whose assembly into a nucleoprotein filament is governed by five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3), which are critical tumor suppressors. Germline mutations in any of these proteins predispose individuals to multiple cancers or debilitating genetic diseases. These paralogs were thought to form two functionally separate complexes, BCDX2 and CX3, that act independently at different stages of HR. Here, we demonstrate that all five paralogs can assemble into a single, ATP-dependent BCDX2-CX3-RAD51 supercomplex. The architecture of this assembly bound to single-stranded DNA (ssDNA) reveals a contiguous filament where the CX3 module stacks atop BCDX2, creating a protofilament template for RAD51 nucleation. We further identify a novel, RAD51B-independent DX2-CX3 complex functioning as a stable RAD51 anchor, and we capture its structure in multiple conformational states, including a post-synaptic state engaged with a DNA bubble. These distinct assemblies are differentially regulated by ATPase activity, defining a dynamic "loader" (BCDX2-CX3) and a stable "anchor" (DX2-CX3) that provide functional modularity to the HR machinery. This work provides a unified, dynamic mechanism for human RAD51 paralog function and delivers an atomic blueprint for interpreting disease-causing mutations.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Christopher M. Rose

PROVIDER: MSV000098718 | MassIVE | Mon Aug 04 11:13:00 BST 2025

REPOSITORIES: MassIVE

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