title: EMSA and single-molecule force spectroscopy study of interactions between Bacillus subtilis single-stranded DNA-binding protein and single-stranded DNA.
abstract: In this article, interactions between Bacillus subtilis single-stranded DNA binding proteins (BsSSB) and single-stranded DNA (ssDNA) were systematically studied. The effect of different molar ratios between BsSSB and ssDNA on their binding modes was first investigated by electrophoretic mobility shift assays (EMSAs). It is found that a high molar ratio of BsSSB to ssDNA can produce BsSSB-ssDNA complexes formed in the mode of two proteins binding one 65-nt (nucleotide) ssDNA whereas a low molar ratio facilitates the formation of BsSSB-ssDNA complexes in the mode of one protein binding one 65-nt ssDNA. Furthermore, two binding modes are in dynamic equilibrium. The unbinding force of BsSSB-ssDNA complexes was measured quantitatively in solutions with different salt concentrations by using AFM-based single-molecule force spectroscopy (SMFS). Our results show that the unbinding force is about 10 pN higher at high salt concentration (0.5 M NaCl) than at low salt concentration (0.1 M NaCl) and the lifetime of BsSSB-ssDNA complexes at high salt concentration is twice as long as that at low salt concentration. These results indicate that more tightly packed BsSSB-ssDNA complexes can form at high salt (0.5 M NaCl) concentration. In addition, the results of EMSA show that ssDNA, which is bound to BsSSB, can dissociate from BsSSB in the presence of the cDNA strand, indicating the dynamic nature of BsSSB-ssDNA interactions.
authors: State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, 130012, PR China.
authors: Wei
authors: W
authors: Zhang
authors: Xiujuan
authors: X
authors: Lü
authors: Wenke
authors: W
authors: Zhang
authors: Jiacong
authors: J
authors: Shen
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D007700:Kinetics; D018625:Microscopy, Atomic Force; D010957:Plasmids; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013057:Spectrum Analysis; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012965:Sodium Chloride