Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA

Abstract

Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.

Figure 1.

(a) Stretching (solid line)–relaxation (dashed line) curves in the absence of protein (black) at a pulling rate of 250 nm/s and in the presence of 30 μM gp2.5 at pulling rates of 250 nm/s (red), 100 nm/s (green), 25 nm/s (blue) and 5 nm/s (light blue) in 10 mM Hepes (pH 7.5), 50 mM Na⁺ (45 mM NaCl and 5 mM NaOH). (b) Stretching (solid line)–relaxation (dashed line) curves in the absence of protein (black) at a pulling rate of 250 nm/s and in the presence of 530 nM gp2.5-Δ26C at pulling rates of 250 nm/s (red), 100 nm/s (green), 25 nm/s (blue) and 5 nm/s (light blue) in 10 mM Hepes (pH 7.5), 100 mM Na⁺ (95 mM NaCl and 5 mM NaOH).

Figure 2.

Measured nonequilibrium DNA melting force, F_k (ν), as a function of pulling rate ν. Data are shown in the absence of protein (black diamond) and in the presence of 10 μM gp2.5 (red square), 20 μM gp2.5 (green triangle), 30 μM gp2.5 (blue circle), 230 nM gp2.5-Δ26C (pink square), 300 nM gp2.5-Δ26C (light green triangle) and 460 nM gp2.5-Δ26C (cyan circle). Linear fits are shown as continuous lines. Each data point is obtained by averaging three or more measurements, and error bars are determined from the standard error. Data is taken in 10 mM Hepes (pH 7.5) and 50 mM Na⁺ (45 mM NaCl and 5 mM NaOH).

Figure 3.

(a) Protein association rate (k_a) as a function of protein concentration for gp2.5 in 5 mM salt (red diamond), 25 mM salt (green triangle) and 50 mM salt (blue circle). (b) Protein association rate (k_a) as a function of protein concentration for gp2.5-Δ26C in 25 mM salt (green triangle), 50 mM salt (blue circle) and 100 mM salt (brown square). Lines are fit to the data using Equation (5). Dashed lines show the three-dimensional (3D) diffusion limit as discussed in the text (48).

Figure 4.

The measured dependence of logarithm of the equilibrium association constants of SSB proteins to ssDNA (K_ss) and dsDNA (K_ds) as a function of logarithm of salt concentration. (a) Equilibrium association constants to ssDNA (solid symbols) and dsDNA (open symbols) for T7 gp2.5 (squares) and T4 gp32 (circles). (b) Equilibrium association constants to ssDNA (solid symbols) and dsDNA (open symbols) for T7 gp2.5 gp2.5-Δ26C (squares) and T4 gp32 C-terminal truncation mutant *I (circles). The linear fits to the data are shown for both proteins as continuous (ssDNA binding) and dashed (dsDNA binding) lines. The error in measurements is shown for all cases. ssDNA-binding results for gp2.5 are taken from Ref. (19), while results for T4 gp32 are taken from Ref. (28).

Figure 5.

The measured free energy of dimer dissociation (gp2.5) or C-terminus dissociation (gp32) as a function of logarithm of salt concentration. Equilibrium association constants of gp2.5 and gp2.5-Δ26C to dsDNA (K_ds, blue open squares and dashed line) and ssDNA (K_ss, blue filled squares and solid line) in 25 and 50 mM Na⁺ buffer were used to determine the values of Δ G_dimer^{ss, ds} directly by using Equation (7), and the interaction per protein, Δ G_dimer^{ss, ds} /2, is shown here. ssDNA results were obtained from Ref. (19). The blue lines are to guide the eye. Note that for the values measured here, Δ G_dimer from Equation (7) of Ref. (19) is equivalent to Δ G_dimer^{ss, ds} /2 from Equation (7) in this work. The analogous calculation was repeated for T4 gp32 C-terminal domain binding to the protein core domain, based on measured T4 gp32 interactions with dsDNA (red open circles and dashed line) and T4 gp32 interactions with ssDNA (red filled circles and solid line), calculated using previously obtained equilibrium binding constants (28). The red lines are fits to the data.