title: C-terminal domain swapping of SSB changes the size of the ssDNA binding site.
abstract: Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, including DNA replication, repair, and recombination, and is therefore essential for cell survival. Bacterial SSB consists of an N-terminal ssDNA-binding/oligomerization domain and a flexible C-terminal protein-protein interaction domain. We characterized the ssDNA-binding properties of Klebsiella pneumoniae SSB (KpSSB), Salmonella enterica Serovar Typhimurium LT2 SSB (StSSB), Pseudomonas aeruginosa PAO1 SSB (PaSSB), and two chimeric KpSSB proteins, namely, KpSSBnStSSBc and KpSSBnPaSSBc. The C-terminal domain of StSSB or PaSSB was exchanged with that of KpSSB through protein chimeragenesis. By using the electrophoretic mobility shift assay, we characterized the stoichiometry of KpSSB, StSSB, PaSSB, KpSSBnStSSBc, and KpSSBnPaSSBc, complexed with a series of ssDNA homopolymers. The binding site sizes were determined to be 26 ± 2, 21 ± 2, 29 ± 2, 21 ± 2, and 29 ± 2 nucleotides (nt), respectively. Comparison of the binding site sizes of KpSSB, KpSSBnStSSBc, and KpSSBnPaSSBc showed that the C-terminal domain swapping of SSB changes the size of the binding site. Our observations suggest that not only the conserved N-terminal domain but also the C-terminal domain of SSB is an important determinant for ssDNA binding.
authors.affiliation: School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan.
authors.forename: Yen-Hua
authors.initials: YH
authors.lastname: Huang
authors.affiliation: School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan ; Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan.
authors.forename: Cheng-Yang
authors.identifier: 0000-0002-0935-7079
authors.initials: CY
authors.lastname: Huang
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics; D012486:Salmonella typhimurium / Q000737:chemistry / Q000235:genetics
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004247:DNA