Conformational Adaptability of Redβ during DNA Annealing and Implications for Its Structural Relationship with Rad52

Single-strand annealing proteins, such as Redβ from λ phage or eukaryotic Rad52, play roles in homologous recombination. Here, we use atomic force microscopy to examine Redβ quaternary structure and Redβ–DNA complexes. In the absence of DNA, Redβ forms a shallow right-handed helix. The presence of single-stranded DNA (ssDNA) disrupts this structure. Upon addition of a second complementary ssDNA, annealing generates a left-handed helix that incorporates 14 Redβ monomers per helical turn, with each Redβ monomer annealing ≈ 11 bp of DNA. The smallest stable annealing intermediate requires 20 bp DNA and two Redβ monomers. Hence, we propose that Redβ promotes base pairing by first increasing the number of transient interactions between ssDNAs. Then, annealing is promoted by the binding of a second Redβ monomer, which nucleates the formation of a stable annealing intermediate. Using threading, we identify sequence similarities between the RecT/Redβ and the Rad52 families, which strengthens previous suggestions, based on similarities of their quaternary structures, that they share a common mode of action. Hence, our findings have implications for a common mechanism of DNA annealing mediated by single-strand annealing proteins including Rad52.