Characterization of a Single-Stranded DNA Binding Protein from Salmonella enterica Serovar Typhimurium LT2

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Salmonella enterica serovar Typhimurium LT2 SSB ( St SSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified St SSB by gel filtration chromatography showed a stable tetramer in solution. In fluorescence titrations, St SSB bound to 21–38 nucleotides (nt) per tetramer depending on the salt concentration. Using EMSA, we characterized the stoichiometry of St SSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 22 ± 1 nt. Furthermore, EMSA results indicated that the dissociation constants of St SSB for the first tetramer were less than that for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding-mode of St SSB is expected to be noncooperative.