Structural characterization of human RPA70N association with DNA damage response proteins.

The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA metabolism, especially DNA damage response. The N-terminal OB domain of the RPA70 subunit (RPA70N) is a major protein-protein interaction element for RPA and binds to more than 20 partner proteins. Previous crystallography studies of RPA70N with p53, DNA2 and PrimPol fragments revealed that RPA70N binds to amphipathic peptides that mimic ssDNA. NMR chemical-shift studies also provided valuable information on the interaction of RPA70N residues with target sequences. However, it is still unclear how RPA70N recognizes and distinguishes such a diverse group of target proteins. Here, we present high-resolution crystal structures of RPA70N in complex with peptides from eight DNA damage response proteins. The structures show that, in addition to the ssDNA mimicry mode of interaction, RPA70N employs multiple ways to bind its partners. Our results advance the mechanistic understanding of RPA70N-mediated recruitment of DNA damage response proteins.

Bre1/RNF20 promotes Rad51-mediated strand exchange and antagonizes the Srs2/FBH1 helicases.

Central to homologous recombination (HR) is the assembly of Rad51 recombinase on single-strand DNA (ssDNA), forming the Rad51-ssDNA filament. How the Rad51 filament is efficiently established and sustained remains partially understood. Here, we find that the yeast ubiquitin ligase Bre1 and its human homolog RNF20, a tumor suppressor, function as recombination mediators, promoting Rad51 filament formation and subsequent reactions via multiple mechanisms independent of their ligase activities. We show that Bre1/RNF20 interacts with Rad51, directs Rad51 to ssDNA, and facilitates Rad51-ssDNA filament assembly and strand exchange in vitro. In parallel, Bre1/RNF20 interacts with the Srs2 or FBH1 helicase to counteract their disrupting effect on the Rad51 filament. We demonstrate that the above functions of Bre1/RNF20 contribute to HR repair in cells in a manner additive to the mediator protein Rad52 in yeast or BRCA2 in human. Thus, Bre1/RNF20 provides an additional layer of mechanism to directly control Rad51 filament dynamics.