Interaction with single-stranded DNA-binding protein modulates Escherichia coli RadD DNA repair activities

The bacterial RadD enzyme is important for multiple genome maintenance pathways, including RecA DNA strand exchange and RecA-independent suppression of DNA crossover template switching. However, much remains unknown about the precise roles of RadD. One potential clue into RadD mechanisms is its direct interaction with the single-stranded DNA binding protein (SSB), which coats single-stranded DNA exposed during genome maintenance reactions in cells. Interaction with SSB stimulates the ATPase activity of RadD. To probe the mechanism and importance of RadD/SSB complex formation, we identified a pocket on RadD that is essential for binding SSB. In a mechanism shared with many other SSB-interacting proteins, RadD uses a hydrophobic pocket framed by basic residues to bind the C-terminal end of SSB. RadD variants that substitute acidic residues for basic residues in the SSB binding site impair RadD/SSB complex formation and eliminate SSB stimulation of RadD ATPase activity in vitro . Mutant E. coli strains carrying charge reversal radD changes display increased sensitivity to DNA damaging agents synergistically with deletions of radA and recG , although the phenotypes of the SSB-binding radD mutants are not as severe a full radD deletion. This suggests that RadD has multiple functions in the cell, with a subset requiring the interaction with SSB. ### Competing Interest Statement The authors have declared no competing interest.