Characterization of a single-stranded DNA-binding protein from Pseudomonas aeruginosa PAO1.
Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as in DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Pseudomonas aeruginosa PAO1 SSB (PaSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified PaSSB by gel filtration chromatography revealed a stable tetramer in solution. In fluorescence titrations, PaSSB bound 22-32 nucleotides (nt) per tetramer depending on salt concentration. Using EMSA, we characterized the stoichiometry of PaSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 29 ± 1 nt. Furthermore, EMSA results indicated that the dissociation constants of PaSSB for the first tetramer were less than those for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding mode of PaSSB is expected to be noncooperative.
Department of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan.
Hau-Chern
HC
Jan
Yen-Ling
YL
Lee
Cheng-Yang
CY
Huang
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics*; D016415:Sequence Alignment
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins