Characterization of a Single-Stranded DNA-Binding Protein from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 |
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Authors: | Hau-Chern Jan Yen-Ling Lee Cheng-Yang Huang |
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Institution: | (1) Department of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan;(2) Department of Medical Research, Chung Shan Medical University Hospital, No. 110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan; |
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Abstract: | 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. |
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