Conformational adaptability of Redbeta during DNA annealing and implications for its structural relationship with Rad52.
Single-strand annealing proteins, such as Redbeta from lambda phage or eukaryotic Rad52, play roles in homologous recombination. Here, we use atomic force microscopy to examine Redbeta quaternary structure and Redbeta-DNA complexes. In the absence of DNA, Redbeta 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 Redbeta monomers per helical turn, with each Redbeta monomer annealing approximately 11 bp of DNA. The smallest stable annealing intermediate requires 20 bp DNA and two Redbeta monomers. Hence, we propose that Redbeta promotes base pairing by first increasing the number of transient interactions between ssDNAs. Then, annealing is promoted by the binding of a second Redbeta monomer, which nucleates the formation of a stable annealing intermediate. Using threading, we identify sequence similarities between the RecT/Redbeta 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.
Erler
Axel
A
Genomics, Biotechnology Center, Technische Universität Dresden, 01307 Dresden, Germany.
Wegmann
Susanne
S
Elie-Caille
Celine
C
Bradshaw
Charles Richard
CR
Maresca
Marcello
M
Seidel
Ralf
R
Habermann
Bianca
B
Muller
Daniel J
DJ
Stewart
A Francis
AF
D020029:Base Pairing; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D020836:Protein Structure, Quaternary; D051721:Rad52 DNA Repair and Recombination Protein / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011995:Recombination, Genetic
D004277:DNA, Single-Stranded; D051721:Rad52 DNA Repair and Recombination Protein