A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology.
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.
Nanomechanics Group, Biotechnology Center, TU Dresden, Dresden, Germany.
Marcel
M
Ander
Department of Genomics, Biotechnology Center, TU Dresden, Dresden, Germany.
Sivaraman
S
Subramaniam
Division of Biophysics, Institute of Resource Ecology, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.
Karim
K
Fahmy
Department of Genomics, Biotechnology Center, TU Dresden, Dresden, Germany.
A Francis
AF
Stewart
Nanomechanics Group, Biotechnology Center, TU Dresden, Dresden, Germany; Cellular Nanoscience, Center for Plant Molecular Biology (ZMBP), Universität Tübingen, Tübingen, Germany.
Erik
E
Schäffer
D010582:Bacteriophage lambda / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D004260:DNA Repair / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008954:Models, Biological; D012689:Sequence Homology, Nucleic Acid; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins