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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 dependence of logarithm of the equilibrium association constants of SSB proteins to ssDNA (Kss) and dsDNA (Kds) 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).
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Figure 4: The measured dependence of logarithm of the equilibrium association constants of SSB proteins to ssDNA (Kss) and dsDNA (Kds) 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).

Mentions: The results of Kds measurement for gp2.5 as a function of salt concentration are shown in Figure 4a along with the Kss data for gp2.5 from our previous work (19). Presented in the same panels are the analogous data for the T4 gp32 protein obtained previously with the same approach (27,28,31,38). The Discussion section below will compare these two representative bacteriophage ssDNA-binding proteins. gp2.5-Δ26C shows stronger and more salt-dependent binding to both ssDNA and dsDNA (compare data points shown by the blue squares in Figure 4a and b) relative to that of gp2.5. Based on these and other (19,24,39) data, we conclude that these differences between gp2.5 and gp2.5-Δ26C are due to the fact that the DNA-binding site of gp2.5 is normally occluded by the CTT of its dimer partner when in solution, and the CTT must dissociate prior to DNA binding, as previously shown (19).Figure 4.


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 dependence of logarithm of the equilibrium association constants of SSB proteins to ssDNA (Kss) and dsDNA (Kds) 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).
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Show All Figures
getmorefigures.php?uid=PMC2553585&req=5

Figure 4: The measured dependence of logarithm of the equilibrium association constants of SSB proteins to ssDNA (Kss) and dsDNA (Kds) 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).
Mentions: The results of Kds measurement for gp2.5 as a function of salt concentration are shown in Figure 4a along with the Kss data for gp2.5 from our previous work (19). Presented in the same panels are the analogous data for the T4 gp32 protein obtained previously with the same approach (27,28,31,38). The Discussion section below will compare these two representative bacteriophage ssDNA-binding proteins. gp2.5-Δ26C shows stronger and more salt-dependent binding to both ssDNA and dsDNA (compare data points shown by the blue squares in Figure 4a and b) relative to that of gp2.5. Based on these and other (19,24,39) data, we conclude that these differences between gp2.5 and gp2.5-Δ26C are due to the fact that the DNA-binding site of gp2.5 is normally occluded by the CTT of its dimer partner when in solution, and the CTT must dissociate prior to DNA binding, as previously shown (19).Figure 4.

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