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.

Characterization of single-stranded DNA-binding protein SsbB fromStaphylococcus aureus: SsbB cannot stimulate PriA helicase

“…21,22 Our crystal structure reveals that the N-terminal DNA-binding domain of SaSsbB structurally resembles PriB, although they signicantly differ in the lengths of b4-and b5-sheets (Fig. 7F).…”

“…Thus, SaSsbB and EcPriB have different functions, and PriB is not a counterpart of SsbB. Considering that the mechanisms of action of primosomes involved in DNA replication restart differ between E. coli 21,22 and Gram-positive 50 was calculated from the titration curves of EMSA by determining the concentration of the protein (tetramers) needed to achieve the midpoint value for input DNA binding. Errors are standard deviations determined by three independent titration experiments.…”

Staphylococcus aureus single-stranded DNA-binding protein SsbA can bind but cannot stimulate PriA helicase

“…Construction of the SaDnaD [ 58 ], SaPriA [ 59 ], Klebsiella pneumoniae SSB (KpSSB) [ 60 ], and tag-free KpSSB [ 61 ] expression plasmids has been reported. The gene encoding SaSsbA (the accession number ACY10277) was amplified by PCR using the genomic DNA of S .…”

“…Purification of the recombinant SaDnaD [ 58 ], SaPriA [ 59 ], KpSSB [ 60 ], and tag-free KpSSB [ 61 ] has been reported. The recombinant SaSsbA and KpSSBc were expressed and purified using the protocol described previously for PriB [ 63 ].…”

“…The recombinant tag-free SaSsbA was expressed and purified using the protocol described previously [ 61 ] for Pseudomonas aeruginosa SSB (PaSSB) and Salmonella enterica serovar Typhimurium LT2 SSB (StSSB) with the following modifications. The cells overexpressing the protein were chilled on ice, harvested by centrifugation, resuspended in Buffer C (20 mM Tris-HCl and 50 mM NaCl, pH 7.9) and disrupted by sonication with ice cooling.…”