Structure and mechanism of the phage T4 recombination mediator protein UvsY

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 113; no. 12; pp. 3275 - 3280
• Main Authors: Gajewski, Stefan, Waddell, Michael Brett, Vaithiyalingam, Sivaraja, Nourse, Amanda, Li, Zhenmei, Woetzel, Nils, Alexander, Nathan, Meiler, Jens, White, Stephen W.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 22.03.2016; National Acad Sciences; National Academy of Sciences, Washington, DC (United States)
• Subjects: 60 APPLIED LIFE SCIENCES; Amino Acid Sequence; BASIC BIOLOGICAL SCIENCES; Biological Sciences; Crystal structure; crystallography; Deoxyribonucleic acid; DNA; DNA architecture; DNA binding; Eukaryotes; homologous recombination; Membrane Proteins - chemistry; Membrane Proteins - physiology; Models, Molecular; Molecular Sequence Data; Phage T4; Protein Conformation; Proteins; Scattering; structural modification; Structure-Activity Relationship; Thermodynamics; Viral Proteins - chemistry; Viral Proteins - physiology; crystallography; DNA architecture; homologous recombination; DNA binding; structural modification
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1519154113

The UvsY recombination mediator protein is critical for efficient homologous recombination in bacteriophage T4 and is the functional analog of the eukaryotic Rad52 protein. During T4 homologous recombination, the UvsX recombinase has to compete with the prebound gp32 single-stranded binding protein for DNA-binding sites and UvsY stimulates this filament nucleation event. We report here the crystal structure of UvsY in four similar open-barrel heptameric assemblies and provide structural and biophysical insights into its function. The UvsY heptamer was confirmed in solution by centrifugation and light scattering, and thermodynamic analyses revealed that the UvsY–ssDNA interaction occurs within the assembly via two distinct binding modes. Using surface plasmon resonance, we also examined the binding of UvsY to both ssDNA and the ssDNA–gp32 complex. These analyses confirmed that ssDNA can bind UvsY and gp32 independently and also as a ternary complex. They also showed that residues located on the rimof the heptamer are required for optimal binding to ssDNA, thus identifying the putative ssDNA-binding surface. We propose a model in which UvsY promotes a helical ssDNA conformation that disfavors the binding of gp32 and initiates the assembly of the ssDNA–UvsX filament.