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Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of Actinomycin D.

Paramanathan T, Vladescu I, McCauley MJ, Rouzina I, Williams MC - Nucleic Acids Res. (2012)

Bottom Line: To resolve this controversy, we develop a method to quantify ActD's equilibrium and kinetic DNA-binding properties as a function of stretching force applied to a single DNA molecule.While we find the preferred ActD-DNA-binding mode to be to two DNA strands, major duplex deformations appear to be a pre-requisite for ActD binding.These results provide quantitative support for a model in which the biologically active mode of ActD binding is to pre-melted dsDNA, as found in transcription bubbles.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Northeastern University, Boston, MA-02115, USA.

ABSTRACT
Actinomycin D (ActD) is a small molecule with strong antibiotic and anticancer activity. However, its biologically relevant DNA-binding mechanism has never been resolved, with some studies suggesting that the primary binding mode is intercalation, and others suggesting that single-stranded DNA binding is most important. To resolve this controversy, we develop a method to quantify ActD's equilibrium and kinetic DNA-binding properties as a function of stretching force applied to a single DNA molecule. We find that destabilization of double stranded DNA (dsDNA) by force exponentially facilitates the extremely slow ActD-dsDNA on and off rates, with a much stronger effect on association, resulting in overall enhancement of equilibrium ActD binding. While we find the preferred ActD-DNA-binding mode to be to two DNA strands, major duplex deformations appear to be a pre-requisite for ActD binding. These results provide quantitative support for a model in which the biologically active mode of ActD binding is to pre-melted dsDNA, as found in transcription bubbles. DNA in transcriptionally hyperactive cancer cells will therefore likely efficiently and rapidly bind low ActD concentrations (≈ 10 nM), essentially locking ActD within dsDNA due to its slow dissociation, blocking RNA synthesis and leading to cell death.

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Relaxation of the DNA extension at constant force in the presence of ActD yields equilibrium binding properties and binding rates. (a) DNA extension as a function of time (open circles) and their exponential fits (lines) at constant force while flowing 500 nM ActD through the flow cell. (b) Force-dependence of the equilibrium dissociation constant, Kd(F) in the presence of 500 nM ActD (green points) and the fit from Equation (4) with Δxeq = 0.20 ± 0.05 nm and Kd(0) = 1.2 ± 0.5 µM (red line). (c) ActD–2DNA on (green points) and off (red points) rates calculated according to Equations (5) and (6), along with directly measured off rates (brown points) from Supplementary Figure S7. The green and red lines are fits to the on and off rates using Equation (7) with the parameters kon(0) = (3.5 ± 0.9) × 10−4/s, koff (0) = (9.8 ± 1.9) × 10−4/s, xon = 0.33 ± 0.03 nm and xoff = 0.11 ± 0.02 nm. (d) Force dependence of the bi-molecular association rate constant ka(F) calculated using measured kon(F) values for 50 nM (blue points), 500 nM (green points) ActD, and concentration-dependent studies at F = 30 pN (pink point) discussed in Supplementary Figure S6. The solid dark red line represents an exponential fit to ka(F) corresponding to Equation (7) with ka(0) = (1.0 ± 0.2) × 103/M s and xon = (0.25 ± 0.02) nm.
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gks069-F5: Relaxation of the DNA extension at constant force in the presence of ActD yields equilibrium binding properties and binding rates. (a) DNA extension as a function of time (open circles) and their exponential fits (lines) at constant force while flowing 500 nM ActD through the flow cell. (b) Force-dependence of the equilibrium dissociation constant, Kd(F) in the presence of 500 nM ActD (green points) and the fit from Equation (4) with Δxeq = 0.20 ± 0.05 nm and Kd(0) = 1.2 ± 0.5 µM (red line). (c) ActD–2DNA on (green points) and off (red points) rates calculated according to Equations (5) and (6), along with directly measured off rates (brown points) from Supplementary Figure S7. The green and red lines are fits to the on and off rates using Equation (7) with the parameters kon(0) = (3.5 ± 0.9) × 10−4/s, koff (0) = (9.8 ± 1.9) × 10−4/s, xon = 0.33 ± 0.03 nm and xoff = 0.11 ± 0.02 nm. (d) Force dependence of the bi-molecular association rate constant ka(F) calculated using measured kon(F) values for 50 nM (blue points), 500 nM (green points) ActD, and concentration-dependent studies at F = 30 pN (pink point) discussed in Supplementary Figure S6. The solid dark red line represents an exponential fit to ka(F) corresponding to Equation (7) with ka(0) = (1.0 ± 0.2) × 103/M s and xon = (0.25 ± 0.02) nm.

Mentions: The non-equilibrium DNA stretch and release curves observed below the melting transition (F < Fm) at 100 nm/s DNA pulling rate are expected to converge at some intermediate equilibrium curve as the DNA pulling rate is decreased to allow more time for ActD–2DNA complex association and dissociation. While we cannot pull slowly enough to directly observe this equilibrium ActD–DNA stretching curve, we can rapidly stretch or stretch and then release DNA to a particular force, and wait for the DNA extension to relax to equilibrium (Figure 4). Confirming our hypothesis, the DNA extension always converges to approximately the same equilibrium value, xeq(F, [ActD]), independent of the stretching history. Following the relaxation of the DNA extension x(t, F, [ActD]) to xeq(F, [ActD]) with time at a constant force allows us to completely characterize the equilibrium affinity and kinetics of ActD–2DNA complex formation (Figure 5). Presented in Figure 5a are four x(t) traces following ActD–DNA association as solution with 500 nM ActD is being flowed through the optical tweezers flow cell (open circles in Figure 5a). Fitting the x(t) traces in Figure 5a to an exponential time dependence (solid curves in Figure 5a) yields the total relaxation rate kt = (kon+koff) of ActD–2DNA and its equilibrium extension per basepair (xeq)(1)Figure 4.


Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of Actinomycin D.

Paramanathan T, Vladescu I, McCauley MJ, Rouzina I, Williams MC - Nucleic Acids Res. (2012)

Relaxation of the DNA extension at constant force in the presence of ActD yields equilibrium binding properties and binding rates. (a) DNA extension as a function of time (open circles) and their exponential fits (lines) at constant force while flowing 500 nM ActD through the flow cell. (b) Force-dependence of the equilibrium dissociation constant, Kd(F) in the presence of 500 nM ActD (green points) and the fit from Equation (4) with Δxeq = 0.20 ± 0.05 nm and Kd(0) = 1.2 ± 0.5 µM (red line). (c) ActD–2DNA on (green points) and off (red points) rates calculated according to Equations (5) and (6), along with directly measured off rates (brown points) from Supplementary Figure S7. The green and red lines are fits to the on and off rates using Equation (7) with the parameters kon(0) = (3.5 ± 0.9) × 10−4/s, koff (0) = (9.8 ± 1.9) × 10−4/s, xon = 0.33 ± 0.03 nm and xoff = 0.11 ± 0.02 nm. (d) Force dependence of the bi-molecular association rate constant ka(F) calculated using measured kon(F) values for 50 nM (blue points), 500 nM (green points) ActD, and concentration-dependent studies at F = 30 pN (pink point) discussed in Supplementary Figure S6. The solid dark red line represents an exponential fit to ka(F) corresponding to Equation (7) with ka(0) = (1.0 ± 0.2) × 103/M s and xon = (0.25 ± 0.02) nm.
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gks069-F5: Relaxation of the DNA extension at constant force in the presence of ActD yields equilibrium binding properties and binding rates. (a) DNA extension as a function of time (open circles) and their exponential fits (lines) at constant force while flowing 500 nM ActD through the flow cell. (b) Force-dependence of the equilibrium dissociation constant, Kd(F) in the presence of 500 nM ActD (green points) and the fit from Equation (4) with Δxeq = 0.20 ± 0.05 nm and Kd(0) = 1.2 ± 0.5 µM (red line). (c) ActD–2DNA on (green points) and off (red points) rates calculated according to Equations (5) and (6), along with directly measured off rates (brown points) from Supplementary Figure S7. The green and red lines are fits to the on and off rates using Equation (7) with the parameters kon(0) = (3.5 ± 0.9) × 10−4/s, koff (0) = (9.8 ± 1.9) × 10−4/s, xon = 0.33 ± 0.03 nm and xoff = 0.11 ± 0.02 nm. (d) Force dependence of the bi-molecular association rate constant ka(F) calculated using measured kon(F) values for 50 nM (blue points), 500 nM (green points) ActD, and concentration-dependent studies at F = 30 pN (pink point) discussed in Supplementary Figure S6. The solid dark red line represents an exponential fit to ka(F) corresponding to Equation (7) with ka(0) = (1.0 ± 0.2) × 103/M s and xon = (0.25 ± 0.02) nm.
Mentions: The non-equilibrium DNA stretch and release curves observed below the melting transition (F < Fm) at 100 nm/s DNA pulling rate are expected to converge at some intermediate equilibrium curve as the DNA pulling rate is decreased to allow more time for ActD–2DNA complex association and dissociation. While we cannot pull slowly enough to directly observe this equilibrium ActD–DNA stretching curve, we can rapidly stretch or stretch and then release DNA to a particular force, and wait for the DNA extension to relax to equilibrium (Figure 4). Confirming our hypothesis, the DNA extension always converges to approximately the same equilibrium value, xeq(F, [ActD]), independent of the stretching history. Following the relaxation of the DNA extension x(t, F, [ActD]) to xeq(F, [ActD]) with time at a constant force allows us to completely characterize the equilibrium affinity and kinetics of ActD–2DNA complex formation (Figure 5). Presented in Figure 5a are four x(t) traces following ActD–DNA association as solution with 500 nM ActD is being flowed through the optical tweezers flow cell (open circles in Figure 5a). Fitting the x(t) traces in Figure 5a to an exponential time dependence (solid curves in Figure 5a) yields the total relaxation rate kt = (kon+koff) of ActD–2DNA and its equilibrium extension per basepair (xeq)(1)Figure 4.

Bottom Line: To resolve this controversy, we develop a method to quantify ActD's equilibrium and kinetic DNA-binding properties as a function of stretching force applied to a single DNA molecule.While we find the preferred ActD-DNA-binding mode to be to two DNA strands, major duplex deformations appear to be a pre-requisite for ActD binding.These results provide quantitative support for a model in which the biologically active mode of ActD binding is to pre-melted dsDNA, as found in transcription bubbles.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Northeastern University, Boston, MA-02115, USA.

ABSTRACT
Actinomycin D (ActD) is a small molecule with strong antibiotic and anticancer activity. However, its biologically relevant DNA-binding mechanism has never been resolved, with some studies suggesting that the primary binding mode is intercalation, and others suggesting that single-stranded DNA binding is most important. To resolve this controversy, we develop a method to quantify ActD's equilibrium and kinetic DNA-binding properties as a function of stretching force applied to a single DNA molecule. We find that destabilization of double stranded DNA (dsDNA) by force exponentially facilitates the extremely slow ActD-dsDNA on and off rates, with a much stronger effect on association, resulting in overall enhancement of equilibrium ActD binding. While we find the preferred ActD-DNA-binding mode to be to two DNA strands, major duplex deformations appear to be a pre-requisite for ActD binding. These results provide quantitative support for a model in which the biologically active mode of ActD binding is to pre-melted dsDNA, as found in transcription bubbles. DNA in transcriptionally hyperactive cancer cells will therefore likely efficiently and rapidly bind low ActD concentrations (≈ 10 nM), essentially locking ActD within dsDNA due to its slow dissociation, blocking RNA synthesis and leading to cell death.

Show MeSH
Related in: MedlinePlus