Crystal structure of the Red C-terminal domain in complex with Exonuclease reveals an unexpected homology with Orf and an interaction with Escherichia coli single stranded DNA binding protein

Bacteriophage encodes a DNA recombination system that includes a 5-3 exonuclease ( Exo) and a single strand annealing protein (Red). The two proteins form a complex that is thought to mediate loading of Red directly onto the single-stranded 3-overhang generated by Exo. Here, we present a 2.3 angstrom crystal structure of the Exo trimer bound to three copies of the Red C-terminal domain (CTD). Mutation of residues at the hydrophobic core of the interface disrupts complex formation in vitro and impairs recombination in vivo. The RedCTD forms a three-helix bundle with unexpected structural homology to phage Orf, a protein that binds to E. coli single-stranded DNA binding protein (SSB) to function as a recombination mediator. Based on this relationship, we found that Red binds to full-length SSB, and to a peptide corresponding to its nine C-terminal residues, in an interaction that requires the CTD. These results suggest a dual role of the CTD, first in binding to Exo to facilitate loading of Reddirectly onto the initial single-stranded DNA (ssDNA) at a 3-overhang, and second in binding to SSB to facilitate annealing of the overhang to SSB-coated ssDNA at the replication fork.