Abstract
Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redβ. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redβ monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redβ clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redβ and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA–protein interaction.
Highlights
In living cells, the genome is constantly being damaged by environmental agents [1]
High-fidelity repair of DNA breaks begins with accurate annealing of the complementary DNA strands
We show that the single-strand annealing protein Redβ initially recognizes sequence homology in the single DNA strands
Summary
The genome is constantly being damaged by environmental agents [1]. Amongst an arsenal of repair pathways, double-strand break repair (DSBR), initiated by single-strand annealing proteins (SSAPs), is central for the maintenance of genomic integrity [2,3]. In addition to a recently detected amino acid signature, SSAPs share several biochemical properties, including weak ssDNA but mostly no double-stranded DNA (dsDNA) binding and oligomerization to higher order, usually ring-like structures [7,8,9,10]. These spectacular structures have been the source of models relating the rings to the promotion of annealing [6,11]. Ring structures have only been seen at high protein concentrations, which have been reported to impair DNA binding and annealing activity [12,13,14,15]. How can homology be detected by proteins that bind DNA in a sequence-independent manner, and how is the fidelity of recombination ensured against false positives?
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