A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology

Marcel Ander; Sivaraman Subramaniam; Karim Fahmy; A Francis Stewart; Erik Schäffer

doi:10.1371/journal.pbio.1002213

A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology

PLoS Biol. 2015 Aug 13;13(8):e1002213. doi: 10.1371/journal.pbio.1002213. eCollection 2015 Aug.

Authors

Marcel Ander¹, Sivaraman Subramaniam², Karim Fahmy³, A Francis Stewart², Erik Schäffer⁴

Affiliations

¹ Nanomechanics Group, Biotechnology Center, TU Dresden, Dresden, Germany.
² Department of Genomics, Biotechnology Center, TU Dresden, Dresden, Germany.
³ Division of Biophysics, Institute of Resource Ecology, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.
⁴ Nanomechanics Group, Biotechnology Center, TU Dresden, Dresden, Germany; Cellular Nanoscience, Center for Plant Molecular Biology (ZMBP), Universität Tübingen, Tübingen, Germany.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacteriophage lambda / genetics*
Bacteriophage lambda / metabolism
Blotting, Western
DNA Repair / physiology*
DNA, Single-Stranded / metabolism*
DNA, Viral / metabolism*
DNA-Binding Proteins / metabolism*
Electrophoresis, Polyacrylamide Gel
Escherichia coli / genetics
Models, Biological
Sequence Homology, Nucleic Acid*
Viral Proteins / metabolism*

Substances

DNA, Single-Stranded
DNA, Viral
DNA-Binding Proteins
Viral Proteins

Grants and funding

MA thanks the German National Academic Foundation for funding. The work was supported by the Deutsche Forschungsgemeinschaft (Emmy Noether Program), European Research Council (ERC Starting Grant 2010, Nanomech 260875), and the Universität Tübingen to ES, and the Technische Universität Dresden Elite University Support the Best Program to AFS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.