Whole Genome Sequencing of SSB IDL mutants

In Escherichia coli, the single-stranded DNA binding protein (SSB) acts as an organizational hub for genome maintenance by interacting with DNA metabolism proteins though the SSB C-terminus. The C-terminus of SSB is comprised of a poorly conserved, intrinsically disordered linker (IDL) and a highly conserved tip (SSB-Ct). SSB-interacting proteins (SIPs) share a conserved mode of SSB recognition, binding to the SSB-Ct. RecG, a helicase implicated in several DNA repair roles, interacts with SSB. A second mode of recognition, where SIPs interact with the IDL of SSB, has been proposed for RecG. To further investigate the RecG-SSB interaction, binding between RecG, SSB, and SSB peptides was measured using fluorescence polarization and isothermal titration calorimetry. We identify the RecG-SSB interaction interface, revealing that RecG binds directly and specifically to the SSB-Ct and not the IDL. Mutating the SSB binding pocket on RecG results in a variant that is functional in vitro but exhibits defects in vivo, including UV sensitivity and SOS induction. The broader role of the SSB IDL is investigated in vivo. Cells expressing SSB IDL deletion variants are viable and not defective in DNA repair. This study reveals that the SSB IDL is not a major contributor to SSB-protein interactions.