Allosteric Effect of E. coli SSB C-Terminal Tails on RecOR Binding to DNA

Abstract

The E. coli single-stranded DNA binding (SSB) protein interacts with at least 15 different proteins, known as SSB-interacting proteins (SIP), during DNA replication, repair, and recombination. However, the specificity by which SSB differentiates and recruits each SIP for different roles is not understood. The E. coli RecO protein is a recombination mediator protein (RMP) involved in the RecF pathway of homologous recombination. RecO forms complexes with RecF and RecR, and is thought to interact with the last 9 amino acids of the intrinsically disordered C-terminal tails of SSB (SSB-Ct). We are examining the interaction of SSB with RecOR by monitoring the change in intrinsic Trp fluorescence of RecO. We observe an allosteric effect of the SSB-Ct on DNA binding by RecOR, such that the SSB-Ct peptide enhances RecOR binding to ssDNA. Whereas structures of RecOR complexes from other organisms, such as D. Radiodurans and T. Tengcongensis, have been determined, they differ in stoichiometry. Furthermore, structures of the E. coli RecOR complex are not available. We therefore are investigating the functional oligomeric state of 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 and the RecR tetramer appears to be the form that binds RecO (supported by NIH GM030498 to TML).