The Escherichia coli PriA helicase specifically recognizes gapped DNA substrates: effect of the two nucleotide-binding sites of the enzyme on the recognition process

Abstract

Energetics and specificity of interactions between the Escherichia coli PriA helicase and the gapped DNAs have been studied, using the quantitative fluorescence titration and analytical ultracentrifugation methods. The gap complex has a surprisingly low minimum total site size, corresponding to approximately 7 nucleotides of the single-stranded DNA (ssDNA), as compared with the site size of approximately 20 nucleotides of the enzyme-ssDNA complex. The dramatic difference in stoichiometries indicates that the enzyme predominantly engages the strong DNA-binding subsite in interactions with the gap and assumes a very different orientation in the gap complex, as compared with the complex with the ssDNA. The helicase binds the ssDNA gaps with 4-5 nucleotides with the highest affinity, which is approximately 3 and approximately 2 orders of magnitude larger than the affinities for the ssDNA and double-stranded DNA, respectively. In the gap complex, the protein does not engage in cooperative interactions with the enzyme predominantly associated with the surrounding dsDNA. Binding of nucleoside triphosphate to the strong and weak nucleotide-binding sites of the helicase eliminates the selectivity of the enzyme for the size of the gap, whereas saturation of both sites with ADP leads to amplified affinity for the ssDNA gap containing 5 nucleotides and engagement of an additional protein area in interactions with the nucleic acid.

FIGURE 1.

a, schematic model of the PriA helicase-ssDNA complex, in the absence of the nucleotide cofactors, in the presence of ATP analogs, or at low ADP concentration (20–22, 27). The strong DNA-binding subsite of the enzyme occludes only 5–8 nucleotides and is located in the center of the enzyme molecule on a protruding domain. However, the enzyme occludes the total site size of ∼20 nucleotides. The yellow ovals symbolize the strong and weak nucleotide-binding sites of the helicase, and the area on the right symbolizes the DNA-binding subsite on the N terminus of the protein, which is closed in the state without ADP bound (27). b, gapped DNA substrates that are used to examine interactions of the PriA helicase with gapped DNAs. The DNA substrates have two dsDNA parts, which are identical in all substrates. The dsDNA parts are separated by the ssDNA of the gap containing 1–10 nucleotides, which are fluorescent, ethenoadenosine residues (eA).

FIGURE 2.

a, fluorescence titrations (λ_ex = 325 nm, λ_em = 410 nm) of the gapped DNA substrate with the ssDNA gap having 4 nucleotides, with the PriA helicase in buffer C (pH 7.0, 10 °C), at three different concentrations of the nucleic acid: 1 × 10⁻⁷ m (■), 3 × 10⁻⁷ m (□), and 6 × 10⁻⁷ m (●). The solid lines are nonlinear least squares fits of the fluorescence titration curves according to the three-site binding model defined by Equations 5–7 (for details, see “Experimental Procedures”). b, the dependence of the relative fluorescence increase, ΔF, upon the degree of binding, Σθ_i, of the PriA DNA complex. The values of Σθ_i have been determined using the quantitative method described under “Experimental Procedures.” The solid straight lines are the limiting slopes of the high and low affinity binding phases. The dashed straight line is an extrapolation of the total average degree of binding to the maximum value of the observed fluorescence increase, ΔF_max = 1.8 ± 0.1, that provides the maximum stoichiometry of 3.0 ± 0.2 of the PriA-gapped DNA complex. The solid line is the computer simulation of the dependence of ΔF as a function of Σθ_i, using the three-site binding model and the obtained binding parameters (Table 1) (for details, see “Experimental Procedures”). c, fluorescence titrations of the gapped DNA substrate with 10 nucleotides in the ssDNA gap with the PriA helicase in buffer C (pH 7.0, 10 °C) at three different concentrations of the nucleic acid: 8.8 × 10⁻⁷ m (■), 2.22 × 10⁻⁶ m (□), and 6 × 10⁻⁷ m (●). The solid lines are nonlinear least squares fits of the fluorescence titration curves according to the three-site binding model defined by Equations 5–7 (for details, see “Experimental Procedures”). d, the dependence of the relative fluorescence increase, ΔF, upon Σθ_i of the PriA-gapped DNA complex. The solid straight line is the limiting slope of the low affinity binding phase. The dashed line is an extrapolation of the total average degree of binding to the maximum value of the observed fluorescence increase ΔF_max = 1.7 ± 0.1 that provides the maximum stoichiometry of 2.8 ± 0.2 of the PriA-gapped DNA complex. The solid line is the computer simulation of the dependence of ΔF as a function of Σθ_i, using the three-site binding model and the obtained binding parameters (Table 1) (for details, see “Experimental Procedures”).

FIGURE 3.

Sedimentation equilibrium concentration profile of the gapped DNA substrate containing 5 nucleotides in the ssDNA gap, labeled at the 5′-end with fluorescein (see “Experimental Procedures”) in the presence of the PriA helicase in buffer C (pH 7.0, 10 °C). The concentrations of the nucleic acid and the protein are 1 × 10⁻⁶ and 2 × 10⁻⁵ m, respectively. The profile has been recorded at 495 nm and at 8000 rpm. The solid line is the nonlinear least squares fit to single exponential function (Equation 4), with a single species having a molecular mass of 242 ± 15 kDa.

FIGURE 4.

The dependence of the logarithm of the binding constant K_G (■) and the logarithm of the binding constant K_DS (□), characterizing the PriA-gapped DNA complex and the association of the PriA helicase with the dsDNA of the gapped DNA substrates, respectively, upon the length of the ssDNA gap, in buffer C (pH 7.0, 10 °C).

FIGURE 5.

a, fluorescence titrations (λ_ex = 325 nm, λ_em = 410 nm) of the gapped DNA substrate having the ssDNA gap with 5 nucleotides (Fig. 1b), with the PriA helicase in buffer C (pH 7.0, 10 °C), containing 1 × 10⁻⁵ m ADP, at two different concentrations of the nucleic acid: 1 × 10⁻⁷ m (■) and 3 × 10⁻⁷ m (□). b, fluorescence titrations (λ_ex = 325 nm, λ_em = 410 nm) of the gapped DNA substrate having the ssDNA gap with 5 nucleotides with the PriA helicase in buffer C (pH 7.0, 10 °C), containing 3 × 10⁻³ m ADP, at two different concentrations of the nucleic acid: 1 × 10⁻⁷ m (■) and 3 × 10⁻⁷ m (□). c, fluorescence titrations (λ_ex = 325 nm, λ_em = 410 nm) of the gapped DNA substrate having the ssDNA gap with 5 nucleotides with the PriA helicase in buffer C (pH 7.0, 10 °C), containing 1 × 10⁻⁵ m ATPγS, at two different concentrations of the nucleic acid: 1 × 10⁻⁷ m (■) and 3 × 10⁻⁷ m (□). d, fluorescence titrations (λ_ex = 325 nm, λ_em = 410 nm) of the gapped DNA substrate having the ssDNA gap with 5 nucleotides with the PriA helicase in buffer C (pH 7.0, 10 °C), containing 3 × 10⁻³ m ATPγS, at two different concentrations of the nucleic acid: 1 × 10⁻⁷ m (■) and 3 × 10⁻⁷ m (□). The solid lines in all panels are nonlinear least squares fits of the fluorescence titration curves according to the three-site binding model defined by Equations 5–7 (for details, see “Experimental Procedures”).

FIGURE 6.

a, the dependence of the logarithm of the binding constant K_G (■) and the logarithm of the binding constant K_DS (□), characterizing the gap complex and the association with the dsDNA, respectively, upon the length of the ssDNA gap in buffer C (pH 7. 0, 10 °C) containing 1 × 10⁻⁵ m ADP. b, the dependence of the logarithm of the binding constant K_G (■) and the logarithm of the binding constant K_DS (□) characterizing the gap complex and the association with the dsDNA, respectively, upon the length of the ssDNA gap in buffer C (pH 7.0, 10 °C) containing 3 × 10⁻³ m ADP. c, the dependence of the logarithm of the binding constant K_G (■) and the logarithm of the binding constant K_DS (□), characterizing the gap complex and the association with the dsDNA, respectively, upon the length of the ssDNA gap in buffer C (pH 7.0, 10 °C) containing 1 × 10⁻⁴ m ATPγS. d, the dependence of the logarithm of the binding constant K_G (■) and the logarithm of the binding constant K_DS (□), characterizing the gap complex and the association with the dsDNA, respectively, upon the length of the ssDNA gap in buffer C (pH 7.0, 10 °C) containing 3 × 10⁻³ m ATPγS. The solid lines in all panels follow the data points and do not have a theoretical basis.

FIGURE 7.

a, fluorescence titrations of the gapped DNA substrate with the ssDNA gap having 5 nucleotides (Fig. 1b), with the PriA helicase (λ_ex = 325 nm, λ_em = 410 nm) in buffer C (pH 7.0, 10 °C), containing different NaCl concentrations: 100 mm (■), 113.5 mm (□), 130 mm (●), 154 mm (○), and 172 mm (▴). The concentration of the gapped DNA substrate is 3 × 10⁻⁷ m. The solid lines are nonlinear least squares fits of the titration curves, using the three-site binding model (Equations 5–7), with σ = 1 and ΔF₁, ΔF_max, K_G, and K_DS of 0.3, 1.5, 1 × 10⁸ m⁻¹, and 2 × 10⁶ m⁻¹ (■); 0.3, 1.33, 3 × 10⁷ m⁻¹, and 6 × 10⁵ m⁻¹ (□); 0.29, 0.95, 9 × 10⁶ m⁻¹, and 4 × 10⁵ m⁻¹ (●); 0.29, 0.77, 7 × 10⁶ m⁻¹, and 3 × 10⁵ m⁻¹ (○); and 0.26, 0.42, 3 × 10⁶ m⁻¹, and 2 × 10⁵ m⁻¹ (▴). b, the dependence of the logarithm of K_G (■) and K_DS (□) upon the logarithm of NaCl. The solid lines are linear least squares fits, which provide the slope ∂logK_G/∂log[NaCl] = −6.1 ± 0.7 and ∂logK_DS/∂log[NaCl] = −3.8 ± 0.5, respectively. c, fluorescence titrations of the gapped DNA substrate with the ssDNA gap having 5 nucleotides (Fig. 1b), with the PriA helicase (λ_ex = 325 nm, λ_em = 410 nm) in buffer C (pH 7.0, 10 °C), containing different MgCl₂ concentrations: 5 mm (■), 7.5 mm (□), 10 mm (●), 12.5 mm (○), and 15 mm (▴). The concentration of the gapped DNA substrate is 3 × 10⁻⁷ m. The solid lines are nonlinear least squares fits of the titration curves, using the three-site binding model (Equations 5–7), with σ = 1 and ΔF₁, ΔF_max, K_G, and K_DS of 0.3, 1.5, 1 × 10⁸ m⁻¹, and 2 × 10⁶ m⁻¹ (■); 0.23, 1.37, 2 × 10⁷ m⁻¹, and 8 × 10⁵ m⁻¹ (□); 0.18, 1.33, 1 × 10⁷ m⁻¹, and 7 × 10⁵ m⁻¹ (●); 0.10, 1.11, 6 × 10⁶ m⁻¹, and 5.1 × 10⁵ m⁻¹ (○); and 0.07, 0.86, 3 × 10⁶ m⁻¹, and 4.5 × 10⁵ m⁻¹ (▴). d, the dependence of the logarithm of K_G (■) and K_DS (□) upon the logarithm of NaCl. The solid lines are linear least squares fits, which provide the slope ∂logK_G/∂log[MgCl₂] = −3.1 ± 0.5 and ∂logK_DS/∂log[MgCl₂] = −1.3 ± 0.3, respectively.

FIGURE 8.

a, fluorescence titrations of the gapped DNA substrate with the ssDNA gap having 5 nucleotides (Fig. 1b), with the PriA helicase (λ_ex = 325 nm, λ_em = 410 nm) in buffer C (pH 7.0, 10 °C), containing 3 × 10⁻³ m ADP and different NaCl concentrations: 100 mm (■), 113.5 mm (□), 133 mm (●), 154 mm (○), and 172 mm (▴). The concentration of the gapped DNA substrate is 3 × 10⁻⁷ m. The solid lines are nonlinear least squares fits of the titration curves, using the three-site binding model (Equations 5–7), with σ = 1 and ΔF₁, ΔF_max, K_G, and K_DS of 0.8, 2.85, 5 × 10⁸ m⁻¹, and 2.5 × 10⁶ m⁻¹ (■); 0.7, 2.55, 1 × 10⁸ m⁻¹, and 1.8 × 10⁶ m⁻¹ (□); 0.70, 2.25, 3 × 10⁷ m⁻¹, and 1.5 × 10⁶ m⁻¹ (●); 0.30, 1.86, 8 × 10⁶ m⁻¹, and 1.1 × 10⁶ m⁻¹ (○); and 0.3, 1.8, 5 × 10⁶ m⁻¹, and 1 × 10⁶ m⁻¹ (▴). b, the dependence of the logarithm of K_G (■) and K_DS (□) upon the logarithm of NaCl. The solid lines are linear least squares fits, which provide the slope ∂logK_G/∂log[NaCl] = −8.5 ± 1.0 and ∂logK_DS/∂log[NaCl] = −1.8 ± 0.4, respectively. c, fluorescence titrations of the gapped DNA substrate with the ssDNA gap having 5 nucleotides, with the PriA helicase (λ_ex = 325 nm, λ_em = 410 nm) in buffer C (pH 7.0, 10 °C), containing 3 × 10⁻³ m ADP and different MgCl₂ concentrations: 5 mm (■), 7.5 mm (□), 10 mm (●), 12.5 mm (○), and 15 mm (▴). The concentration of the gapped DNA substrate is 3 × 10⁻⁷ m. The solid lines are nonlinear least squares fits of the titration curves, using the three-site binding model (Equations 5–7), with σ = 1 and ΔF₁, ΔF_max, K_G, and K_DS of 0.8, 2.85, 5 × 10⁸ m⁻¹, and 2.5 × 10⁶ m⁻¹ (■); 0.65, 2.38, 3 × 10⁸ m⁻¹, and 2.2 × 10⁶ m⁻¹ (□); 0.4, 1.87, 1.5 × 10⁸ m⁻¹, and 1.8 × 10⁶ m⁻¹ (●); 0.25, 1.67, 8 × 10⁷ m⁻¹, and 1.5 × 10⁶ m⁻¹ (○); and 0.1, 1.26, 5 × 10⁷ m⁻¹, and 1.1 × 10⁶ m⁻¹ (▴). d, the dependence of the logarithm of K_G (■) and K_DS (□) upon the logarithm of NaCl. The solid lines are linear least square fits, which provide the slope ∂logK_G/∂log[MgCl₂] = −2.1 ± 0.5 and ∂logK_DS/∂log[MgCl₂] = −0.7 ± 0.2, respectively.

FIGURE 9.

a, schematic model of the PriA complex with the gapped DNA substrate, containing an ssDNA gap with 5 nucleotides, in the absence of the nucleotide cofactors, based on the results obtained in this work. The enzyme binds the gapped DNA substrate using its strong DNA-binding subsite (20–22, 27). In the complex, the ssDNA/dsDNA junctions are predominantly engaged in interactions with the helicase. These interactions match the geometry and structural flexibility of the strong DNA-binding subsite and lead to the significantly amplified affinity for the gapped DNA substrate. b, schematic model of the PriA complex with the gapped DNA substrate, containing an ssDNA gap with 5 nucleotides, with ADP saturating both nucleotide-binding sites of the enzyme. The helicase is bound to the gapped DNA substrate through its strong DNA-binding subsite. However, saturation of both nucleotide-binding sites with ADP induces conformational transition of the enzyme and opens an additional interacting area on the N-terminal domain, which engages an additional fragment of the gapped DNA substrate, analogously to the complexes with the ssDNA. As a result, the enzyme has a further increased affinity for the gapped DNA substrate, as compared with the gap complex formed in the absence of ADP (for details, see “Experimental Procedures”).