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

Shokri L, Marintcheva B, Eldib M, Hanke A, Rouzina I, Williams MC - Nucleic Acids Res. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Northeastern University, 111 Dana Research Center, Boston, MA 02115, USA.

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.

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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 (Kds, blue open squares and dashed line) and ssDNA (Kss, blue filled squares and solid line) in 25 and 50 mM Na+ buffer were used to determine the values of Δ Gdimerss, ds directly by using Equation (7), and the interaction per protein, Δ Gdimerss, 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 Gdimer from Equation (7) of Ref. (19) is equivalent to Δ Gdimerss, 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.
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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 (Kds, blue open squares and dashed line) and ssDNA (Kss, blue filled squares and solid line) in 25 and 50 mM Na+ buffer were used to determine the values of Δ Gdimerss, ds directly by using Equation (7), and the interaction per protein, Δ Gdimerss, 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 Gdimer from Equation (7) of Ref. (19) is equivalent to Δ Gdimerss, 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.

Mentions: The importance and generality of this ‘electrostatic shielding mechanism’ was discussed in the recent study (24). However, in contrast to gp32, the gp2.5 protein forms homodimers in solution (5), while it binds DNA as a monomer. Moreover, it is known that the CTT deletion mutant does not dimerize (39). These observations reflect the current well-supported model that the CTT of gp2.5 binds not to its own DNA-binding site, but rather to the site of its partner, thereby stabilizing the homodimer. We can use our gp2.5 and gp2.5-Δ26C dsDNA- and ssDNA-binding data to estimate the maximum free energy of this protein dimerization, assuming that the DNA binding of the two dimerized gp2.5 proteins differs from that of the two gp2.5-Δ26C proteins by the probability of the thermal dissociation of the gp2.5 dimer, Pdimer = e(− Δ Gdimer /kBT) − (e− Δ Gdimer / kBT + 1), i.e.6Here ΔGdimer is the dimer dissociation free energy, which can be expressed as7Presented in Figure 5 is the dimerization free energy per gp2.5 monomer protein, Δ Gdimer /2, as a function of solution ionic strength estimated from our dsDNA-binding data obtained in this work, as well as from the data for ssDNA binding from our previous work on gp2.5 (19). Both estimates are in good agreement. As expected, the dimerization free energy is salt dependent. This implies that the electrostatic interaction of the anionic CTT with the DNA cationic-binding site of the protein partner contributes significantly to dimerization.Figure 5.


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

Shokri L, Marintcheva B, Eldib M, Hanke A, Rouzina I, Williams MC - Nucleic Acids Res. (2008)

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 (Kds, blue open squares and dashed line) and ssDNA (Kss, blue filled squares and solid line) in 25 and 50 mM Na+ buffer were used to determine the values of Δ Gdimerss, ds directly by using Equation (7), and the interaction per protein, Δ Gdimerss, 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 Gdimer from Equation (7) of Ref. (19) is equivalent to Δ Gdimerss, 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.
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Related In: Results  -  Collection

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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 (Kds, blue open squares and dashed line) and ssDNA (Kss, blue filled squares and solid line) in 25 and 50 mM Na+ buffer were used to determine the values of Δ Gdimerss, ds directly by using Equation (7), and the interaction per protein, Δ Gdimerss, 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 Gdimer from Equation (7) of Ref. (19) is equivalent to Δ Gdimerss, 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.
Mentions: The importance and generality of this ‘electrostatic shielding mechanism’ was discussed in the recent study (24). However, in contrast to gp32, the gp2.5 protein forms homodimers in solution (5), while it binds DNA as a monomer. Moreover, it is known that the CTT deletion mutant does not dimerize (39). These observations reflect the current well-supported model that the CTT of gp2.5 binds not to its own DNA-binding site, but rather to the site of its partner, thereby stabilizing the homodimer. We can use our gp2.5 and gp2.5-Δ26C dsDNA- and ssDNA-binding data to estimate the maximum free energy of this protein dimerization, assuming that the DNA binding of the two dimerized gp2.5 proteins differs from that of the two gp2.5-Δ26C proteins by the probability of the thermal dissociation of the gp2.5 dimer, Pdimer = e(− Δ Gdimer /kBT) − (e− Δ Gdimer / kBT + 1), i.e.6Here ΔGdimer is the dimer dissociation free energy, which can be expressed as7Presented in Figure 5 is the dimerization free energy per gp2.5 monomer protein, Δ Gdimer /2, as a function of solution ionic strength estimated from our dsDNA-binding data obtained in this work, as well as from the data for ssDNA binding from our previous work on gp2.5 (19). Both estimates are in good agreement. As expected, the dimerization free energy is salt dependent. This implies that the electrostatic interaction of the anionic CTT with the DNA cationic-binding site of the protein partner contributes significantly to dimerization.Figure 5.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Northeastern University, 111 Dana Research Center, Boston, MA 02115, USA.

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.

Show MeSH
Related in: MedlinePlus