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.

Figure 1

Construction of plasmids for expression of the chimeric KpSSBnStSSBc and KpSSBnPaSSBc proteins. To investigate the effect of the C-terminal domain of SSB on the size of the ssDNA-binding site, the C-terminal domain of KpSSB was replaced by that of StSSB and PaSSB. pET21b-KpSSB (Primers 7 and 8), pET21b-StSSB (Primers 9 and 10), and pET21b-PaSSB (Primers 11 and 12) vectors were mutated to create a desired SacI site and to obtain the vectors for expression of the chimeric proteins KpSSBnStSSBc and KpSSBnPaSSBc. The D91E/Q92L-engineered pET21b-KpSSB vector, the D91E/Q92L-engineered pET21b-StSSB vector, and the G90E/Q91L-engineered pET21b-PaSSB vector were cut at NdeI and SacI sites. Subsequently, the KpSSBn, StSSBc-pET21b, and PaSSBc-pET21b fragments were purified. KpSSBn was ligated with StSSBc-pET21b and PaSSBc-pET21b fragments to generate the engineered pET21b-KpSSBnStSSBc and pET21b-KpSSBnPaSSBc vectors. To avoid artificial residues, positions 91 and 92 of the two plasmids were mutated back (Primers 13 to 16) to obtain pET21b-KpSSBnStSSBc and pET21b-KpSSBnPaSSBc vectors. Thus, pET21b-KpSSBnStSSBc and pET21b-KpSSBnPaSSBc will express KpSSB1-91 fused StSSB92-176 and PaSSB91-165, respectively. Note that KpSSBnPaSSBc will have 166 amino acid residues.

Figure 2

Multiple amino acid sequence alignment of SSB proteins. Sequence alignment of KpSSB, StSSB, and PaSSB was generated by CLUSTALW2. Identical amino acid residues are colored in red. Gly and Gln residues are shaded in cyan and gray. The N-terminal domains of these SSBs are significantly conserved.

Figure 3

Protein purity. Coomassie Blue-stained SDS-PAGE (15%) of the purified KpSSB (lane 1), StSSB (lane 2), PaSSB (lane 3), KpSSBnStSSBc (lane 4), KpSSBnPaSSBc (lane 5), and molecular mass standards (M) are shown. The sizes of the standard proteins, from the top down, are as follows: 55, 40, 35, 25, 15, and 10 kDa. The purified SSBs migrated between the 25 and 15 kDa standards on the SDS-PAGE.

Figure 4

Binding of KpSSB to dT20–60. KpSSB (0, 19, 37, 77, 155, 310, 630, 1250, 2500, and 5000 nM) was incubated for 30 min at 25°C with 1.7 nM of (a) dT20, (b) dT25, (c) dT35, (d) dT45, (e) dT50, (f) dT55, or (g) dT60 in a total volume of 10 μL in 20 mM Tris-HCl pH 8.0 and 100 mM NaCl. Aliquots (5 μL) were removed from each reaction solution and added to 2 μL of gel-loading solution (0.25% bromophenol blue and 40% sucrose). The resulting samples were resolved on a native 8% polyacrylamide gel at 4°C in TBE buffer (89 mM Tris borate and 1 mM EDTA) for 1 h at 100 V and visualized by autoradiography. Complexed and free DNA bands were scanned and quantified.

Figure 5

Binding of StSSB to dT15–50. StSSB (0, 19, 37, 77, 155, 310, 630, 1250, 2500, and 5000 nM) was incubated for 30 min at 25°C with 1.7 nM of (a) dT15, (b) dT20, (c) dT30, (d) dT40, (e) dT45, or (f) dT50 in a total volume of 10 μL in 20 mM Tris-HCl pH 8.0 and 100 mM NaCl. Aliquots (5 μL) were removed from each reaction solution and added to 2 μL of gel-loading solution (0.25% bromophenol blue and 40% sucrose). The resulting samples were resolved on a native 8% polyacrylamide gel at 4°C in TBE buffer (89 mM Tris borate and 1 mM EDTA) for 1 h at 100 V and visualized by autoradiography. Complexed and free DNA bands were scanned and quantified.

Figure 6

Binding of PaSSB to dT20–65. PaSSB (0, 19, 37, 77, 155, 310, 630, 1250, 2500, and 5000 nM) was incubated for 30 min at 25°C with 1.7 nM of (a) dT20, (b) dT25, (c) dT35, (d) dT45, (e) dT55, (f) dT60, or (g) dT65 in a total volume of 10 μL in 20 mM Tris-HCl pH 8.0 and 100 mM NaCl. Aliquots (5 μL) were removed from each reaction solution and added to 2 μL of gel-loading solution (0.25% bromophenol blue and 40% sucrose). The resulting samples were resolved on a native 8% polyacrylamide gel at 4°C in TBE buffer (89 mM Tris borate and 1 mM EDTA) for 1 h at 100 V and visualized by autoradiography. Complexed and free DNA bands were scanned and quantified.

Figure 7

Binding of KpSSBnStSSBc to dT15–45. KpSSBnStSSBc (0, 19, 37, 77, 155, 310, 630, 1250, 2500, and 5000 nM) was incubated for 30 min at 25°C with 1.7 nM of (a) dT15, (b) dT20, (c) dT40, or (d) dT45 in a total volume of 10 μL in 20 mM Tris-HCl pH 8.0 and 100 mM NaCl. Aliquots (5 μL) were removed from each reaction solution and added to 2 μL of gel-loading solution (0.25% bromophenol blue and 40% sucrose). The resulting samples were resolved on a native 8% polyacrylamide gel at 4°C in TBE buffer (89 mM Tris borate and 1 mM EDTA) for 1 h at 100 V and visualized by autoradiography. Complexed and free DNA bands were scanned and quantified.

Figure 8

Binding of KpSSBnPaSSBc to dT20–60. KpSSBnPaSSBc (0, 19, 37, 77, 155, 310, 630, 1250, 2500, and 5000 nM) was incubated for 30 min at 25°C with 1.7 nM of (a) dT20, (b) dT25, (c) dT40, (d) dT55, or (e) dT60 in a total volume of 10 μL in 20 mM Tris-HCl pH 8.0 and 100 mM NaCl. Aliquots (5 μL) were removed from each reaction solution and added to 2 μL of gel-loading solution (0.25% bromophenol blue and 40% sucrose). The resulting samples were resolved on a native 8% polyacrylamide gel at 4°C in TBE buffer (89 mM Tris borate and 1 mM EDTA) for 1 h at 100 V and visualized by autoradiography. Complexed and free DNA bands were scanned and quantified.

Figure 9

Gel-filtration chromatographic analyses of KpSSBnStSSBc and KpSSBnPaSSBc. Purified protein (2 mg/mL) was applied to a Superdex 200 HR 10/30 column (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) equilibrated with Buffer D. The column was operated at a flow rate of 0.5 mL/min, and 0.5 mL fractions were collected. The proteins were detected by measuring the absorbance at 280 nm. The column was calibrated with proteins of known molecular weight: thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa). The K _av values for the standard proteins and the SSB variants were calculated from the equation: K _av = (V _e − V _o)/(V _c − V _o), where V _o is column void volume, V _e is elution volume, and V _c is geometric column volume.

Figure 10

Structure modeling of SSB. The structures of KpSSB1–115, StSSB1–115, and PaSSB1–115 (the N-terminal domain of SSB) were modeled by SWISS-MODEL. The structures of KpSSB116–142, StSSB116–142, and PaSSB121–160 (the C-terminal domain of SSB) were modeled by (PS)². Other regions of SSBs could not be modeled by these two programs. The structures of the N-terminal domain and the C-terminal domain of these SSBs were manually linked (KpSSB1–142, blue; StSSB1–142, pink; PaSSB1–160, green) and superimposed with the crystal structure of EcSSB1–142 (orange) (PDB entry: 1QVC) for comparison. For clarity, only one subunit of the tetramer was shown for each SSB.

Figure 11

Possible models for explaining why SSBs are with different binding site sizes. Two modeled structures of KpSSB1–142 (blue), StSSB1–142 (pink), and PaSSB1–160 (green) complexed with ssDNA (gold) are shown. For clarity, only one C-terminal domain was shown for each SSB tetramer. By using the electrophoretic mobility shift assay and the protein chimeragenesis, we characterized that the binding site sizes of KpSSB, StSSB, PaSSB, KpSSBnStSSBc, and KpSSBnPaSSBc were 26, 21, 29, 21, and 29 nt per tetramer, respectively. KpSSB, StSSB, and PaSSB are similar proteins whose N-terminal ssDNA-binding domains are almost identical. Thus, the C-terminal domain of SSB may indirectly contribute to ssDNA binding and wrapping and affects the binding site size by the steric hindrance.