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 directly interacts with the E. coli ssDNA binding protein (EcoSSB). Analytical ultracentrifugation shows that all 4 carboxy-termini of an EcoSSB tetramer bind exonuclease I. Binding is weakened by increasing salt concentrations, indicating the involvement of the negatively charged amino acids of the carboxy-terminus of SSB. Mutant SSB proteins EcoSSBP176S (ssb-113) and EcoSSBF177C do not bindtoexonuclease I while EcoSSBG15D (ssb-3) does bind. In a co-precipitation assay we show that the absence of the lastten amino acids (PMDFDDDIPF) completely abolishes binding of EcoSSB to exonuclease I. The interaction does not depend on the presence of the correct amino-terminal DNA binding domain or the amino acid sequences between the DNA binding domain and the last ten amino acids. A synthetic peptide (WMDFDDDIPF), corresponding to the last nine amino acids of EcoSSB, specifically inhibits the interaction. Both EcoSSBP176S and EcoSSBF177C SSBs bind DNA similar to wild-type EcoSSB, indicating that the phenotype of ssb-113 is not an indication of altered DNA binding. The repair deficiency of either ssb-3 or ssb-113 strain can be complemented by overexpression of the respective other mutant.
Genschel
J
J
Medizinische Hochschule Hannover, Biophysikalische Chemie, Hannover, Germany.
Curth
U
U
Urbanke
C
C
D001665:Binding Sites; D011232:Chemical Precipitation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D007700:Kinetics; D009994:Osmolar Concentration; D011071:Poly T / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D011067:Poly dA-dT / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D011071:Poly T; D011067:Poly dA-dT; D011072:Poly U; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I