The Role of Intrinsically Disordered Linker and Salt Type on Cooperativity of E. Coli SSB Binding to ssDNA

E. coli single strand DNA binding protein (SSB) is a key protein in DNA replication, recombination and repair. SSB functions as a homotetramer and binds ssDNA in different modes that differ in their excluded site size and cooperative binding to ssDNA. Two types of cooperativity have been identified previously: the high “unlimited”, nearest-neighbor type of cooperativity associated with the 35 site size ((SSB)35) binding mode at low [NaCl], and a lower, “limited” cooperativity associated with formation of dimers of tetramers in the 65 site size ((SSB)65) binding mode at [NaCl] > 200 mM. Using sedimentation velocity and equilibrium titration methods we have examined how salt concentration and type affects these cooperativities and binding mode transitions on long ssDNA, focusing on the effects of KGlu, since glutamate is the major monovalent anion in E. coli. Unexpectedly, we find that in contrast to NaCl, KGlu promotes highly cooperative binding to ssDNA even at high concentrations of 0.5 M, conditions that promote the ((SSB)65) mode, suggesting that highly cooperative binding is likely occurring in vivo. We also show that all cooperative binding behavior absolutely requires the unstructured C-terminal region (intrinsically disordered linker (IDL)) connecting the SSB DNA binding core and the 9 amino acid C-terminal end (tip) needed for SSB binding to other proteins involved in genome maintenance (supported by NIH GM030498 to TML).