Domain Structure of the Redo 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

Red is a component of the Red recombination system of bacteriophage A that promotes a single strand annealing (SSA) reaction to generate end-to-end concatemers of the phage genome for packaging. Red beta interacts with A exonuclease (lambda exo), 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 Red beta 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 (Red beta(FL)) and the N-terminal domain (Red beta(177)) to different lengths of ssDNA substrate and annealed duplex product. We find that in general, Red beta(FL) binds more tightly to annealed duplex product than to ssDNA substrate, while Red beta(177) binds more tightly to ssDNA. In addition, the C-terminal region of Red beta corresponding to residues 182-261 was purified and found to fold into an a-helical domain that is required for the interaction with lambda exo to form the synaptosome complex. Deletion analysis of Red beta revealed that removal of just eleven residues from the C-terminus disrupts the interaction with lambda exo as well as ssDNA and dsDNA recombination in vivo. By contrast, the determinants for self-oligomerization of Red beta appear to reside solely within the N-terminal domain. The subtle but significant differences in the relative binding of Red beta(FL) and Red beta(177) to ssDNA substrate and annealed duplex product may be important for Red beta to function as a SSA protein in vivo. (C) 2016 Elsevier Ltd. All rights reserved.