Domain Structure of the Redβ Single-Strand Annealing Protein: the C-terminal Domain is Required for Fine-Tuning DNA-binding Properties, Interaction with the Exonuclease Partner, and Recombination in vivo
Christopher E. Smith, Charles E. Bell
Journal of Molecular Biology
Redβ is a component of the Red recombination system of bacteriophage λ that promotes a single strand annealing (SSA) reaction to generate end-to-end concatemers of the phage genome for packaging. Redβ interacts with λ exonuclease (λexo), the other component of the Red system, to form a “synaptosome” complex that somehow integrates the end resection and annealing steps of the reaction. Previous work using limited proteolysis and chemical modification revealed that Redβ consists of an N-terminal DNA binding domain, residues 1–177, and a flexible C-terminal “tail”, residues 178–261. Here, we quantitatively compare the binding of the full-length protein (RedβFL) and the N-terminal domain (Redβ177) to different lengths of ssDNA substrate and annealed duplex product. We find that in general, RedβFL binds more tightly to annealed duplex product than to ssDNA substrate, while Redβ177 binds more tightly to ssDNA. In addition, the C-terminal region of Redβ corresponding to residues 182–261 was purified and found to fold into an α-helical domain that is required for the interaction with λexo to form the synaptosome complex. Deletion analysis of Redβ revealed that removal of just eleven residues from the C-terminus disrupts the interaction with λexo as well as ssDNA and dsDNA recombination in vivo. By contrast, the determinants for self-oligomerization of Redβ appear to reside solely within the N-terminal domain. The subtle but significant differences in the relative binding of RedβFL and Redβ177 to ssDNA substrate and annealed duplex product may be important for Redβ to function as a SSA protein in vivo.
Circular dichroism, Ligand binding, Protein folding, Biochemistry