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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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Measured nonequilibrium DNA melting force, Fk (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).
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Figure 2: Measured nonequilibrium DNA melting force, Fk (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).

Mentions: We used DNA stretching to probe the effect of gp2.5 and gp2.5-Δ26C on the DNA melting force as a function of pulling rate. Stretching curves for a single λ-DNA molecule in the absence or presence of gp2.5 and gp2.5-Δ26C are shown in Figure 1a and b, respectively. In both cases, the presence of the protein reduces the DNA melting force. However, to observe considerable reduction in the overstretching force, higher concentrations of gp2.5 compared to gp2.5-Δ26C are required. As the dsDNA molecule is pulled at different rates of v = 5–250 nm/s, the molecule extends to the B-form contour length and then begins to melt at the particular force Fk (v) (where the subscript k indicates that this kinetically determined force is likely to depend on pulling rate v) (27,28). In the absence of protein, the DNA melting force is independent of the pulling rate and shows very little hysteresis. However, in the presence of both gp2.5 and gp2.5-Δ26C, the melting force is significantly lowered and moreover depends on the pulling rate. The hysteresis observed in the release part of the stretching cycle clearly demonstrates the nonequilibrium nature of the DNA melting by gp2.5 and its C-terminal deletion mutant. The observed nonequilibrium DNA melting force is determined by the rate of protein binding to ssDNA during duplex melting. Therefore, this force is analog to dsDNA thermal melting studies and different from the equilibrium DNA melting force that was used in our previous studies (19). However, while in thermal melting studies, the DNA melting temperature varies linearly with the logarithm of the heating rate (29,30), in nonequilibrium DNA force-induced melting, the melting force varies linearly with the logarithm of the pulling rate [this work, Figure 2, and (27,28)].Figure 1.


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)

Measured nonequilibrium DNA melting force, Fk (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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553585&req=5

Figure 2: Measured nonequilibrium DNA melting force, Fk (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).
Mentions: We used DNA stretching to probe the effect of gp2.5 and gp2.5-Δ26C on the DNA melting force as a function of pulling rate. Stretching curves for a single λ-DNA molecule in the absence or presence of gp2.5 and gp2.5-Δ26C are shown in Figure 1a and b, respectively. In both cases, the presence of the protein reduces the DNA melting force. However, to observe considerable reduction in the overstretching force, higher concentrations of gp2.5 compared to gp2.5-Δ26C are required. As the dsDNA molecule is pulled at different rates of v = 5–250 nm/s, the molecule extends to the B-form contour length and then begins to melt at the particular force Fk (v) (where the subscript k indicates that this kinetically determined force is likely to depend on pulling rate v) (27,28). In the absence of protein, the DNA melting force is independent of the pulling rate and shows very little hysteresis. However, in the presence of both gp2.5 and gp2.5-Δ26C, the melting force is significantly lowered and moreover depends on the pulling rate. The hysteresis observed in the release part of the stretching cycle clearly demonstrates the nonequilibrium nature of the DNA melting by gp2.5 and its C-terminal deletion mutant. The observed nonequilibrium DNA melting force is determined by the rate of protein binding to ssDNA during duplex melting. Therefore, this force is analog to dsDNA thermal melting studies and different from the equilibrium DNA melting force that was used in our previous studies (19). However, while in thermal melting studies, the DNA melting temperature varies linearly with the logarithm of the heating rate (29,30), in nonequilibrium DNA force-induced melting, the melting force varies linearly with the logarithm of the pulling rate [this work, Figure 2, and (27,28)].Figure 1.

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