Local structural dynamics of Rad51 protomers revealed by cryo-electron microscopy of Rad51-ssDNA filaments.

Homologous recombination (HR) is a high-fidelity repair mechanism for double-strand breaks. Rad51 is the key enzyme that forms filaments on single-stranded DNA (ssDNA) to catalyze homology search and DNA strand exchange in recombinational DNA repair. In this study, we employed single-particle cryo-electron microscopy (cryo-EM) to ascertain the density map of the budding yeast Rad51-ssDNA filament bound to ADP-AlF 3 , 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-AlF 3 . It shows 6-fold symmetry implying monomeric building blocks, unlike the structure of the Rad51-I345T mutant filament with three-fold symmetry implying dimeric building blocks, for which the structural comparisons provide a satisfying mechanistic explanation. This image analysis enables comprehensive comparisons of individual Rad51 protomers within the filament and reveals local conformational movements of amino acid side chains. Notably, Arg293 in Loop1 adopts multiple conformations to facilitate Leu296 and Val331 in separating and twisting the DNA triplets. We also analyzed the predicted structures of yeast Rad51-K342E and two tumor-derived human RAD51 variants, RAD51-Q268P and RAD51-Q272L, using the Rad51-ssDNA structure from this study as a reference.