The homotetrameric E. coli single stranded DNA binding (SSB) protein plays a central role in DNA replication, repair and recombination. E. coli SSB can bind to long single-stranded (ss) DNA in multiple binding modes using all four subunits ((SSB)65 mode) or only two subunits ((SSB)35 binding mode), with the binding mode preference regulated by salt concentration and SSB binding density. These binding modes display very different ssDNA binding properties with the (SSB)35 mode displaying highly cooperative binding to ssDNA. SSB tetramers also bind an array of partner proteins, recruiting them to their sites of action. This is achieved through interactions with the last 9 amino acids (acidic tip) of the intrinsically disordered linkers (IDLs) within the four C-terminal tails connected to the ssDNA binding domains. Here we show that the amino acid composition and length of the IDL affects the ssDNA binding mode preferences of SSB protein. Surprisingly the number of IDLs and the lengths of individual IDLs together with the acidic tip contribute to highly cooperative binding in the (SSB)35 binding mode. Hydrodynamic studies and atomistic simulations suggest that the E. coli SSB IDLs show a preference for forming an ensemble of globular conformations, whereas the IDL from Plasmodium falciparum SSB forms an ensemble of more extended random coils. The more globular conformations correlate with cooperative binding.

E. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds to ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes that differ in occluded site size and cooperativity. High “unlimited” cooperativity is associated with the 35 site size ((SSB)35) mode at low [NaCl], whereas the 65 site size ((SSB)65) mode formed at higher [NaCl] (> 200 mM), where ssDNA wraps completely around the tetramer, displays “limited” cooperativity forming dimers of tetramers. It was previously thought that high cooperativity was associated only with the (SSB)35 binding mode. However, we show here that highly cooperative binding also occurs in the (SSB)65 binding mode at physiological salt concentrations containing either glutamate or acetate. Highly cooperative binding requires the 56 amino acid intrinsically disordered C-terminal linker (IDL) that connects the DNA binding domain with the 9 amino acid C-terminal acidic tip that is involved in SSB binding to other proteins involved in genome maintenance. These results suggest that high cooperativity involves interactions between IDL regions from different SSB tetramers. Glutamate, which is preferentially excluded from protein surfaces, may generally promote interactions between intrinsically disordered regions of proteins. Since glutamate is the major monovalent anion in E. coli, these results suggest that SSB likely binds to ssDNA with high cooperativity in vivo.

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Background: RecBCD helicase is involved in repair of double-stranded DNA breaks. Results: The 5Ј to 3Ј ssDNA translocation rate of RecBCD is faster than the 3Ј to 5Ј rate in the absence of a CHI site, and the rates are coupled asymmetrically. Conclusion: RecBC controls 3Ј to 5Ј and 5Ј to 3Ј translocation, but RecD controls only 5Ј to 3Ј translocation. Significance: Asymmetric regulation may explain how RecBCD is regulated after CHI recognition.

E. coli single strand DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function we engineered covalently linked forms of SSB that possess only one or two C-termini within a four OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents, but accumulate more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.