The Escherichia coli PriA helicase-double-stranded DNA complex: location of the strong DNA-binding subsite on the helicase domain of the protein and the affinity control by the two nucleotide-binding sites of the enzyme

Abstract

The Escherichia coli PriA helicase complex with the double-stranded DNA (dsDNA), the location of the strong DNA-binding subsite, and the effect of the nucleotide cofactors, bound to the strong and weak nucleotide-binding site of the enzyme on the dsDNA affinity, have been analyzed using the fluorescence titration, analytical ultracentrifugation, and photo-cross-linking techniques. The total site size of the PriA-dsDNA complex is only 5±1 bp, that is, dramatically lower than 20±3 nucleotides occluded in the enzyme-single-stranded DNA (ssDNA) complex. The helicase associates with the dsDNA using its strong ssDNA-binding subsite in an orientation very different from the complex with the ssDNA. The strong DNA-binding subsite of the enzyme is located on the helicase domain of the PriA protein. The dsDNA intrinsic affinity is considerably higher than the ssDNA affinity and the binding process is accompanied by a significant positive cooperativity. Association of cofactors with strong and weak nucleotide-binding sites of the protein profoundly affects the intrinsic affinity and the cooperativity, without affecting the stoichiometry. ATP analog binding to either site diminishes the intrinsic affinity but preserves the cooperativity. ADP binding to the strong site leads to a dramatic increase of the cooperativity and only slightly affects the affinity, while saturation of both sites with ADP strongly increases the affinity and eliminates the cooperativity. Thus, the coordinated action of both nucleotide-binding sites on the PriA-dsDNA interactions depends on the structure of the phosphate group. The significance of these results for the enzyme activities in recognizing primosome assembly sites or the ssDNA gaps is discussed.

Fig. 1

(a) Schematic model of the PriA helicase–ssDNA complex, in the absence of nucleotide cofactors, in the presence of ATP analogs, or at low ADP concentration.^– The strong DNA-binding subsite of the enzyme, which encompasses only 5–7 nucleotides, is located in the center of the enzyme molecule on a protruding domain. Data obtained in this work show that the strong DNA-binding site is located on the helicase domain of the enzyme (see above). The enzyme occludes the total site size of ~20 nucleotides. Yellow ovals symbolize the strong and weak nucleotide-binding sites of the helicase. (b) Schematic model of the PriA helicase complex with the ssDNA in the presence of high concentrations of ADP, which saturates both nucleotide-binding sites of the enzyme. The helicase, bound to the ssDNA through its strong DNA-binding subsite, engages an additional fragment of the nucleic acid, presumably through the binding subsite located on the N-terminal domain of the enzyme.^–

Fig. 2

(a) Fluorescence titrations of the dsDNA 10-mer, labeled at the 5′ end of one of the strands with fluorescein (Materials and Methods), with the PriA protein (λ_ex =485 nm, λ_em =520 nm) in buffer C (pH 7.0, 10 °C), containing 50 mM NaCl, in the absence of nucleotide cofactors, at two different nucleic acid concentrations: ■, 1×10⁻⁸ M; □, 5×10⁻⁸ M (oligomer). The continuous lines are nonlinear least-squares fits of the titration curves, using the statistical thermodynamic model described by Eqs. (1)–(3) (Table 1). (b) Dependence of the relative fluorescence increase of the dsDNA 10-mer, ΔF, upon the total average degree of binding, ΣΘ_i (■). The continuous lines indicate the limiting slopes of the plot at the low and high enzyme concentration range, respectively, and have no theoretical basis. The broken line is the extrapolation of ΔF to the maximum value of ΔF_max =0.41.

Fig. 3

(a) Fluorescence titrations of the dsDNA 15-mer, labeled at the 5′ end of one of the strands with fluorescein (Materials and Methods), with the PriA protein (λ_ex = 485 nm, λ_em =520 nm) in buffer C (pH 7.0, 10 °C), containing 50 mM NaCl, in the absence of nucleotide cofactors, at two different nucleic acid concentrations: ■, 1×10⁻⁸ M; □, 5×10⁻⁸ M (oligomer). The continuous lines are nonlinear least-squares fits of the titration curves, using the statistical thermodynamic model described by Eqs. (4)–(7). (b) Dependence of the relative fluorescence increase of the dsDNA 15-mer, ΔF, upon the total average degree of binding, ΣΘ_i (■). The continuous line is the computer simulation of the relative fluorescence increase of the dsDNA 15-mer upon the total average degree of binding of the PriA helicase on the nucleic acid, obtained using the determined intrinsic binding constant K₁₅ =(1± 0.4) × 10⁷ M⁻¹, the cooperativity factor ω=8, and spectroscopic parameters ΔF₁ =0.09, ΔF₂ =0.43, and ΔF₃ =0.19.

Fig. 4

Sedimentation equilibrium concentration profile of the dsDNA 10-mer, labeled at the 5′ end with fluorescein (Materials and Methods) in the presence of PriA helicase in buffer C (pH 7.0, 10 °C). The concentrations of the nucleic acid and the helicase are 2.6×10⁻⁷ M and 8×10⁻⁶ M, respectively. The profile has been recorded at 495 nm and at 8000 rpm. The continuous line is the nonlinear least-squares fit to single exponential function [Eq. (22)], with a single species having a molecular weight of 156,000±15,000.

Fig. 5

Fluorescence titrations of the fluorescent reference ssDNA 18-mer, dεA(pεA)₁₇, with the PriA protein (λ_ex =325 nm, λ_em =410 nm) in buffer C (pH 7.0, 10 °C) in the absence (■) and presence of two different concentrations of the unmodified dsDNA 10-mer (Materials and Methods). The concentration of the reference ssDNA 18-mer is 1×10⁻⁶ M. The concentrations of the unmodified dsDNA 10-mer are 1.5×10⁻⁷ M (□) and 5×10⁻⁷ M (●). The continuous lines are nonlinear least-squares fits of the binding titration curves using Eqs. (8)–(11) (lattice competition titrations) with the binding constant for dεA (pεA)₁₇, K₁₈ =1.6×10⁶ M⁻¹ and ΔF_max =1.67, and the intrinsic binding constant, K_10S =8×10⁶ M⁻¹ and ω_S =6, for the unmodified dsDNA 10-mer.

Fig. 6

(a) Fluorescence titrations of the dsDNA 10-mer, labeled at the 5′ end of one of the strands with fluorescein (Materials and Methods), with the PriA protein (λ_ex = 485 nm, λ_em =520 nm) in buffer C (pH 7.0, 10 °C), containing different NaCl concentrations: 50 mM (■), 69 mM (□), 88 mM (●), 107 mM (○), 126 mM (◆). The continuous lines are nonlinear least-squares fits of the titration curves, using the statistical thermodynamic model described by Eqs. (1)–(3), with ΔF₁ =0.124 and ΔF_max =0.4, and K₁₀ and ω, respectively, as follows: 1.6×10⁷ M⁻¹ and 10 (■), 7.0×10⁶ M⁻¹ and 10 (□), 4.0×10⁶ M⁻¹ and 8 (●), 2.0×10⁶ M⁻¹ and 6 (○), 8.0×10⁵ M⁻¹ and 5 (◆). (b) The dependence of the logarithm of the intrinsic binding constant, K₁₀, upon the logarithm of NaCl concentration. The continuous line is a linear least-squares fit, which provides the slope ∂logK₁₀/∂log[NaCl]= −3.1±0.6. (c) The dependence of the logarithm of the cooperativity parameter, ω, upon the logarithm of NaCl concentration. The continuous line is a linear least-squares fit, which provides the slope ∂logω/∂log[NaCl]=−1±0.3.

Fig. 7

(a) Fluorescence titrations of the dsDNA 10-mer, labeled with fluorescein at the 5′ end (Materials and Methods), with the PriA helicase in buffer C (pH 7.0), at different temperatures (°C): 5 (△), 7.5 (○), 10 (■), 15 (□), and 20 (●). The continuous lines are nonlinear least-squares fits of the titration curves, using the statistical thermodynamic model described by Eqs. (1)–(3). The concentration of the DNA substrate is 1.5×10⁻⁸ M. (b) The dependence of the natural logarithm of the intrinsic binding constant, K₁₀, upon the reciprocal of the temperature (Kelvin) (van’t Hoff plot). The continuous line is the nonlinear least-squares fit of the experimental plot to the model of binding of two PriA molecules in two different conformations, defined by Eqs. (15)–(18) (see the text for details). The dependence of the natural logarithm of the cooperativity parameter, ω, upon the reciprocal of temperature (Kelvin). The continuous line is a linear least-squares fit, which provides the slope ΔH_ω=−28.9 kcal/mol.

Fig. 8

(a) Fluorescence titrations of the dsDNA 10-mer, labeled at the 5′ end with fluorescein (Materials and Methods) with the PriA helicase in buffer C (pH 7.0, 10 °C), containing 1 × 10⁻⁵ M ADP, at two different nucleic acid concentrations: ■, 1 × 10⁻⁸ M; □, 2.5 × 10⁻⁸ M (oligomer). The continuous lines are nonlinear least-squares fits of the titration curves, using the statistical thermodynamic model described by Eqs. (1)–(3) (Table 1). (b) Dependence of the relative fluorescence increase of the dsDNA 10-mer, ΔF, upon the total average degree of binding of PriA helicase on the nucleic acid (■). The continuous line follows the experimental points and has no theoretical basis. The broken line is the extrapolation of ΔF to ΔF_max = 0.29.

Fig. 9

(a) Fluorescence titrations of the dsDNA 10-mer, labeled at the 5′ end with fluorescein (Materials and Methods) with the PriA helicase in buffer C (pH 7.0, 10 °C), containing 3 × 10⁻³ M ADP, at two different nucleic acid concentrations: ■, 1 × 10⁻⁸ M; □, 2.5 × 10⁻⁸ M (oligomer). The continuous lines are nonlinear least-squares fits of the titration curves, using the statistical thermodynamic model described by Eqs. (1)–(3) (Table 1). (b) Dependence of the relative fluorescence increase of the dsDNA 10-mer, ΔF, upon the average number of bound PriA molecules (■). The continuous lines indicate the limiting slopes of the plot at low and high enzyme concentration ranges, respectively, and have no theoretical basis. The broken line is the extrapolation of ΔF to the maximum value of ΔF_max = 0.93.

Fig. 10

Fluorescence titrations of the dsDNA 10-mer, labeled at the 5′ end with fluorescein (Materials and Methods) with the PriA helicase in buffer C (pH 7.0, 10 °C), containing 1 × 10⁻⁵ M (○) and 3 × 10⁻³ M (●) ATPγS, respectively. For comparison, the titrations of the same nucleic acid, in the presence of 1 × 10⁻⁵ M (□) and 3 × 10⁻³ M (■) ADP, are also included in the figure. The concentration of the nucleic acid is 2.5 × 10⁻⁸ M (oligomer). The continuous lines are nonlinear least-squares fits of the titration curves, using the statistical thermodynamic model described by Eqs. (1)–(3) (Table 1).

Fig. 11

(a) The SDS polyacrylamide gel (10%) of the PriA protein–[³²P]-dT₂₀ complex subjected to the time-dependent trypsin digestion and stained with Coomassie Brilliant Blue (Materials and Methods). Lane 0 contains the molecular markers; lane 1 contains 5′ [³²P]-dT₂₀ alone without irradiation; lane 2 contains 5′ [³²P]-dT₂₀ alone after irradiation; lane 3 contains the PriA protein–[³²P]-dT₂₀ complex in the absence of the protease prior to irradiation; lane 4 contains the PriA protein–[³²P]-dT₂₀ complex, in the absence of the protease after irradiation; lanes 5 to 11 contain the PriA protein–[³²P]-dT₂₀ complex in the presence of the constant trypsin concentration, collected at different time intervals of the digestion reaction: lane 5, 1 min; lane 6, 3 min; lane 7, 5 min; lane 8, 10 min; lane 9, 20 min; lane 10, 30 min. (b) Autoradiogram of the same 10% SDS polyacrylamide gel of the PriA protein–dT(pT)₁₉ complex, as shown in (a) (see the text for details).

Fig. 12

Schematic functional models of the PAS-like structures and the ssDNA gap recognition processes by the PriA helicase–nucleotide cofactor complexes. In the case of the PAS-like structure recognition (a), binding of ADP to the strong nucleotide-binding site, or hydrolysis of the bound ATP and the release of ADP from the weak nucleotide-binding site, induces a significant positive cooperativity, which reinforces the enzyme affinity for the dsDNA of the PAS structure. As a result, more than one PriA molecule may participate in the initial PAS recognition process. With both nucleotide-binding sites saturated with ADP, the positive cooperativity is strongly diminished, but the intrinsic affinity of the enzyme is strongly increased, preserving the specificity of the recognition of the PAS structure. In the case of the ssDNA gap at the stalled replication fork (b), mostly the complex, with both nucleotide-binding sites of the PriA protein saturated with ADP, seems to be involved, although the enzyme molecule without nucleotide cofactors may also participate in the recognition process. Low cooperativity of the PriA protein binding to the DNA, with two nucleotide-binding sites saturated with ADP, indicates that a single PriA molecule participates in the ssDNA gap recognition (see the text for details).