Assembly and Binding of E. coli RecO and Record Proteins to SSB-Ct Tails

The E. coli RecO protein is a recombination mediator protein (RMP) involved in the RecF pathway of homologous recombination, and it is one of at least 15 different proteins that are known to interact with the last 9 amino acids of the intrinsically disordred C-terminal tails ofE. coli single-stranded DNA binding (SSB) protein during DNA replication, recombination, and repair. Whereas structures of RecOR complexes from other organisms have been determined, they show different RecO/RecR stoichiometries. Furthermore, crystal structures of the E. coli RecOR complex are not available. We therefore are investigating the functional oligomeric states of E. coli RecO and RecR, and the stoichiometry of the RecOR complex using analytical ultracentrifugation and isothermal titration calorimetry. E. coli RecR had previously been reported to form a dimer, but we find that it is in a dimer-tetramer equilibrium that is sensitive to pH with lower pH stabilizing the tetrameric species. In fact, we find that the RecR tetramer is the form that binds RecO with a minimal stoichiometry of one RecO per RecR tetramer. We have also examined the effect on RecO-RecR interactions of the binding of RecO to the intrinsically disordered SSB tail.