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

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Abstract

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 (StSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified StSSB by gel filtration chromatography showed a stable tetramer in solution. In fluorescence titrations, StSSB bound to 21–38 nucleotides (nt) per tetramer depending on the salt concentration. Using EMSA, we characterized the stoichiometry of StSSB 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 StSSB for the first tetramer were less than that for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding-mode of StSSB is expected to be noncooperative.

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Abbreviations

Ec :

Escherichia coli

St :

Salmonella enterica serovar Typhimurium LT2

Pa :

Pseudomonas aeruginosa PAO1

Mt :

Mycobacterium tuberculosis

Ms :

Mycobacterium smegmatis

Hp :

Helicobacter pylori

ssDNA:

Single-stranded DNA

SSB:

Single-stranded DNA-binding protein

SDS-PAGE:

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis

EDTA:

Ethylenediamine tetraacetic acid

EMSA:

Electrophoretic mobility shift analysis

nt:

Nucleotides

K d :

The apparent dissociation constant

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Acknowledgments

We thank Ms. Hui-Chuan Hsieh for constructing the pET21b-StSSB. This research was supported a grant from the National Research Program for Genome Medicine (NSC 99-3112-B-040-001 to C.Y. Huang).

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Authors and Affiliations

  1. Department of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan

    Yen-Hua Huang, Yen-Ling Lee & Cheng-Yang Huang

  2. Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan

    Cheng-Yang Huang

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  1. Yen-Hua Huang
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  2. Yen-Ling Lee
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Correspondence to Cheng-Yang Huang.

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Huang, YH., Lee, YL. & Huang, CY. Characterization of a Single-Stranded DNA Binding Protein from Salmonella enterica Serovar Typhimurium LT2. Protein J 30, 102–108 (2011). https://doi.org/10.1007/s10930-011-9309-1

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