Mechanism of Rad51 filament formation by Rad52 and Rad55-Rad57 in homologous recombination

ABSTRACT: Homologous recombination (HR) repairs double-stranded DNA breaks (DSBs). The DSBs are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Rad51 is a recombinase and catalyzes strand invasion and the search for homology. However, it binds to ssDNA with lower affinity than RPA. Thus, mediator proteins such as Rad52 (yeast) or BRCA2 (humans) are required to promote Rad51 binding to RPA-coated ssDNA, but the underlying mechanisms remain poorly understood. Saccharomyces cerevisiae Rad52 interacts with Rad51 through two distinct binding modes. We here uncover that the Rad51-binding site in the disordered C-terminus of Rad52 sorts polydisperse Rad51 into discrete monomers. We developed fluorescent-Rad51 to directly visualize filament formation in single molecule optical tweezer analysis and capture Rad52 catalyzed Rad51 loading onto RPA-coated ssDNA with a distinct preference for junctions. Addition of the Rad51-paralog Rad55-Rad57 increases the number of Rad51 bound by ~60%. Deletion of the C-terminus of Rad52 results in loss of Rad51 sorting and abrogates Rad51 binding to RPA-coated DNA. While BRCA2 and Rad52 are structurally unrelated, we show that many of the functional features are conserved. We describe a concerted Sort, Stack & Extend (SSE) mechanism for mediator-paralog catalyzed Rad51 filament formation in HR.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Monika Tokmina-Lukaszewska  

LAB HEAD: Edwin Antony

PROVIDER: PXD065573 | Pride | 2025-07-28

REPOSITORIES: Pride