Regulation of Nearest-Neighbor Cooperative Binding of E. coli SSB Protein to DNA

Escherichia coli single-strand (ss) DNA-binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes differing in occluded site size and cooperativity. The 35-site-size ((SSB)₃₅) mode favored at low [NaCl] and high SSB/DNA ratios displays high "unlimited" nearest-neighbor cooperativity (ω₃₅), forming long protein clusters, whereas the 65-site-size ((SSB)₆₅) mode in which ssDNA wraps completely around the tetramer is favored at higher [NaCl] (>200 mM) and displays "limited" cooperativity (ω₆₅), forming only dimers of tetramers. In addition, a non-nearest-neighbor high cooperativity can also occur in the (SSB)₆₅ mode on long ssDNA even at physiological salt concentrations in the presence of glutamate and requires its intrinsically disordered C-terminal linker (IDL) region. However, whether cooperativity exists between the different modes and the role of the IDL in nearest-neighbor cooperativity has not been probed. Here, we combine sedimentation velocity and fluorescence titration studies to examine nearest-neighbor cooperativity in each binding mode and between binding modes using (dT)₇₀ and (dT)₁₄₀. We find that the (SSB)₃₅ mode always shows extremely high "unlimited" cooperativity that requires the IDL. At high salt, wild-type SSB and a variant without the IDL, SSB-ΔL, bind in the (SSB)₆₅ mode but show little cooperativity, although cooperativity increases at lower [NaCl] for wild-type SSB. We also find significant intermode nearest-neighbor cooperativity (ω_65/35), with ω₆₅ ≪ ω_65/35 <ω₃₅. The intrinsically disordered region of SSB is required for all cooperative interactions; however, in contrast to the non-nearest-neighbor cooperativity observed on longer ssDNA, glutamate does not enhance these nearest-neighbor cooperativities. Therefore, we show that SSB possesses four types of cooperative interactions, with clear differences in the forces stabilizing nearest-neighbor versus non-nearest-neighbor cooperativity.