Interaction of E-coli single-stranded DNA binding protein (SSB) with exonuclease I. The carboxy-terminus of SSB is the recognition site for the nuclease

The 3'-5' single-stranded DNA (ssDNA) degrading exonuclease I of E. coli di rectly interacts with the E. coli ssDNA binding protein (EcoSSB). Analytica l ultracentrifugation shows that all 4 carboxy-termini of an EcoSSB tetrame r bind exonuclease I. Binding is weakened by increasing salt concentrations , indicating the involvement of the negatively charged amino acids of the c arboxy-terminus of SSB. Mutant SSB proteins EcoSSBP176S (ssb-113) and EcoSS BF177C do not bind to exonuclease I white EcoSSBG15D (ssb-3) does bind. In a co-precipitation assay we show that the absence of the last ten amino aci ds (PMDFDDDIPF) completely abolishes binding of EcoSSB to exonuclease I. Th e interaction does not depend on the presence of the correct amino-terminal DNA binding domain or the amino acid sequences between the DNA binding dom ain and the last ten amino acids. A synthetic peptide (WMDFDDDIPF), corresp onding to the last nine amino acids of EcoSSB, specifically inhibits the in teraction. Both EcoSSBP176S and EcoSSBF177C SSBs bind DNA similar to wild-t ype EcoSSB, indicating that the phenotype of ssb-113 is not an indication o f altered DNA binding. The repair deficiency of either ssb-3 or ssb-113 str ain can be complemented by overexpression of the respective other mutant.