Abstract
Redβ is a 261 amino acid protein from bacteriophage λ that promotes a single-strand annealing (SSA) reaction for repair of double-stranded DNA (dsDNA) breaks. While there is currently no high-resolution structure available for Redβ, models of its DNA binding domain (residues 1–188) have been proposed based on homology with human Rad52, and a crystal structure of its C-terminal domain (CTD, residues 193-261), which binds to λ exonuclease and E. coli single-stranded DNA binding protein (SSB), has been determined. To evaluate these models, the 14 lysine residues of Redβ were mutated to alanine, and the variants tested for recombination in vivo and DNA binding and annealing in vitro. Most of the lysines within the DNA binding domain, including K36, K61, K111, K132, K148, K154, and K172, were found to be critical for DNA binding in vitro and recombination in vivo. By contrast, none of the lysines within the CTD, including K214, K245, K251, K253, and K258 were required for DNA binding in vitro, but two, K214 and K253, were critical for recombination in vivo, likely due to their involvement in binding to SSB. K61 was identified as a residue that is critical for DNA annealing, but not for initial ssDNA binding, suggesting a role in binding to the second strand of DNA incorporated into the complex. The K148A variant, which has previously been shown to be defective in oligomer formation, had the lowest affinity for ssDNA, and was the only variant that was completely non-cooperative, suggesting that ssDNA binding is coupled to oligomerization.
Highlights
Proteins that bind to single-stranded DNA and promote the annealing of complementary strands are called single-strand annealing (SSA) proteins [1]
There is no high-resolution structure of full-length Redβ, a crystal structure of the CTD has been determined [26] and models for the N-terminal DNAbinding domain have been proposed based on distant homology with Rad52 (Figure 1 and Figure S2) [14,15,16]
Low resolution EM images of Redβ have led to a compelling model in which the protein binds to initial single-stranded DNA (ssDNA) substrate as an oligomeric ring, and transitions to a helical filament when a second strand of complementary ssDNA is added to form the complex with annealed duplex [32,33]
Summary
Proteins that bind to single-stranded DNA (ssDNA) and promote the annealing of complementary strands are called single-strand annealing (SSA) proteins [1] They typically have important roles in DNA recombination, most notably in the SSA pathway for repair of double-stranded DNA (dsDNA) breaks [2], and in the second end capture step of homology directed repair and homologous recombination [3]. At least four different families of SSA proteins have been identified, based on their distinct 3-dimensional folds. The authors suggest that this site may have a role in bringing together multiple ring-ssDNA complexes to promote annealing They propose a mechanism in which ssDNA is initially bound to the inner site, but moves to the outer site as the annealing reaction proceeds [13]
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