Mechanism of homologous recombination: Structure and assembly of the Rad51 filament by Rad51 paralogs

Homologous recombination (HR) is an important DNA repair pathway that safeguards genome integrity. During HR, the Rad51 nucleoprotein filament catalyzes strand invasion into a homologous duplex DNA. We employed single-particle cryogenic electron microscopy (cryo-EM) to ascertain the density map of the wild type budding yeast Rad51-ssDNA filament bound to ADP-AlF3, achieving a resolution of 2.35 Å without imposing helical symmetry. The model assigned 6 Rad51 protomers, 24 nt of DNA, and 6 bound ADP-AlF3. It shows 6-fold symmetry implying monomeric building blocks and enables comprehensive comparisons of individual Rad51 protomers within the filament and revealing local conformational movements of amino acid side chains. Rad51-ssDNA filament formation requires a conserved family of Rad51 paralogs that act as tumor suppressors in humans. By capturing six distinct states using cryo-electron microscopy, we reveal that the yeast Rad51 paralog complex, composed of the Rad55-Rad57 heterodimer and the SHU (Psy3-Csm2-Shu1-Shu2) complex, selectively brings Rad51 to single-stranded DNA to seed filament formation. Rad51 is itself a transient yet integral component of this machinery, binding along the Rad57 subunit to complete a high-affinity DNA-binding site. We also uncover a dual-nucleotide regulatory mechanism: a structural ADP molecule stabilizes the complex, while a second, catalytic ATPase site at the Rad57-Rad51 interface promotes release of the paralog complex. These structural and mechanistic features provide a blueprint for understanding the function of Rad51 paralogs across eukaryotes.