Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded 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 accumulates 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. [Display omitted] •We have made covalently linked SSB proteins containing four OB-folds that differ in the number of C-terminal tails.•Linked SSBs with two C-tails are functional in vivo and can couple leading and lagging strand DNA replication in vitro.•Linked SSBs with two C-tails show a higher rate of spontaneous mutagenesis. The SSB protein couples DNA repair and replication through its C-terminal tails.•Linked SSBs with one tail are dominant negative and show a defect in coupling leading and lagging strand DNA replication in vitro.•Linked SSBs with one tail are also defective in supporting replication restart.