Joudeh, L. | Search bioRxiv

bioRxiv2025

Structural mechanism of strand exchange by the RAD51 filament.

Homologous Recombination (HR) preserves genomic stability by repairing double-strand DNA breaks and ensuring efficient DNA replication. Central to HR is the strand-exchange reaction taking place within the three-stranded synapsis wherein a RAD51 nucleoprotein filament binds to a donor DNA. Here we present the cryoEM structure of a displacement loop of human RAD51 that captures the synaptic state when the filament has become tightly bound to the donor DNA. The structure elucidates the mechanism of strand exchange by RAD51, including the filament engagement with the donor DNA, the strand invasion and pairing with the complementary sequence of the donor DNA, the capture of the non-complementary strand and the polarity of the strand-exchange reaction. Our findings provide fundamental mechanistic insights into the biochemical reaction of eukaryotic HR.

molecular biology↗

bioRxiv2023

A metal ion-dependent mechanism of RAD51 nucleoprotein filament disassembly

The RAD51 ATPase polymerises on single-stranded DNA to form nucleoprotein filaments (NPFs) that are critical intermediates in the DNA strand-exchange reactions of Homologous Recombination (HR). ATP binding is important to maintain the NPF in a competent conformation for strand pairing and exchange. Once strand exchange is completed, ATP hydrolysis licenses the filament for disassembly. Here we show using high-resolution cryoEM that the ATP-binding site of the RAD51 NPF contains a second metal ion. In the presence of ATP, the metal ion promotes the local folding of RAD51 into the conformation required for DNA binding. The metal ion is absent in the structure of an ADP-bound RAD51 filament, that rearranges in a conformation incompatible with DNA binding. The presence of the second metal ion explains how RAD51 couples the nucleotide state of the filament to DNA binding. We propose that loss of the second metal ion upon ATP hydrolysis drives RAD51 dissociation from the DNA and weakens filament stability, thus contributing to NPF disassembly, a critical step in the completion of HR.

biochemistry↗