BRCA2 and RAD54B FxPP motifs Bind DMC1 Filaments through Persistent and Transient Interfaces

Abstract

During meiosis, double-strand DNA breaks are programmed to initiate homologous recombination. The DMC1 recombinogenic filament is central to the repair of these breaks. However, the three-dimensional structures of the protein-protein interfaces regulating the assembly and activity of this filament have not been described yet. We and others previously reported that in BRCA2, a P-motif called PhePP binds to DMC1 in its oligomeric and filament states. Here we identified a similar P-motif in the DNA translocase RAD54B. We solved the cryo-electron microscopy (cryo-EM) structures of BRCA2 and RAD54B P-motif peptides bound to a ssDNA-DMC1 filament at 1.9-2.0 [A] resolution. While these peptides only share the sequence F-[IV]-P-P, they bind to the filament through larger 9-10 residue core sequences with superimposable structures. Both peptides bind to a hydrophobic and negatively charged site, named the P-site, on a single DMC1 protomer and stabilize the ssDNA-DMC1 filament. Mutagenesis experiments and molecular dynamics simulations identified additional transient interactions between positively charged residues of the peptides and negatively charged patches distributed on the DMC1 protomers. We propose that BRCA2 and RAD54B stably dock at the P-site of a single DMC1 protomer but also bridge two DMC1 protomers within the presynaptic filament via transient contacts with the adjacent protomer.