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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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Actinomycin D structure and DNA interactions. (a) Chemical structure of ActinomycinD (ActD), with the planar phenoxazone ring system shown in green and pentapeptide side chains shown in red. (b) Ball and stick structure of two ActD molecules interacting with two DNA strands (different shades of blue) obtained from the pdb file IMNV, where phenoxazone rings (cyan for top molecule and green for bottom molecule) intercalate between DNA base pairs and the pentapeptide side chains (red) lie in the minor groove.
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gks069-F1: Actinomycin D structure and DNA interactions. (a) Chemical structure of ActinomycinD (ActD), with the planar phenoxazone ring system shown in green and pentapeptide side chains shown in red. (b) Ball and stick structure of two ActD molecules interacting with two DNA strands (different shades of blue) obtained from the pdb file IMNV, where phenoxazone rings (cyan for top molecule and green for bottom molecule) intercalate between DNA base pairs and the pentapeptide side chains (red) lie in the minor groove.

Mentions: Actinomycin D (ActD) is a DNA binding (1) small molecule with potent activity as an antibiotic (2) and anticancer agent (3). It is a neutral molecule that contains a planar tricyclic phenoxazone ring that intercalates dsDNA and two cyclic pentapeptide side chains (Figure 1a). ActD can intercalate between double stranded DNA (dsDNA) base pairs (4–8), bind to single-stranded DNA (ssDNA) (9–12) and can even ‘hemi-intercalate’ between the bases of a single DNA strand (13,14). Early studies found that once bound ActD dissociates slowly from dsDNA (4), with a component of its dissociation occurring on a time scale of ∼1000 s. These studies attributed ActD’s anticancer activity to this slow kinetics, and found it to be due to the slow fitting of its two highly stressed cyclic penta-peptide side chains into the DNA minor groove below and above the intercalated phenoxazone ring (4,15) (Figure 1b). The fitting into the groove is stabilized by hydrogen bonding of the ActD side chains to guanine bases (5–7), and associated with major DNA duplex deformations, such as strong bending (6,8), unwinding (6,16) and even base flipping (16,17). Duplex deformations are also driven by optimization of the tricyclic phenoxazone ring stacking with the 3′ faces of guanine (or adenine) residues in the opposite DNA strands (8,14,16). Competing models for the anticancer activity of ActD depend on the favored binding mode; Intercalation may inhibit replication by stabilizing dsDNA in front of the replication fork (8), while binding to destabilized duplexes such as transcription bubbles may inhibit DNA transcription (18–20), and ssDNA binding may directly stall the DNA polymerase (12). However, despite many years of study by a variety of methods and detailed knowledge of the relationship between DNA sequence, structure and the strength of ActD–DNA interactions, there is no consensus for any of these models and the reason for the selective anti-cancer activity of ActD at low concentrations remains unclear.Figure 1.


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)

Actinomycin D structure and DNA interactions. (a) Chemical structure of ActinomycinD (ActD), with the planar phenoxazone ring system shown in green and pentapeptide side chains shown in red. (b) Ball and stick structure of two ActD molecules interacting with two DNA strands (different shades of blue) obtained from the pdb file IMNV, where phenoxazone rings (cyan for top molecule and green for bottom molecule) intercalate between DNA base pairs and the pentapeptide side chains (red) lie in the minor groove.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367174&req=5

gks069-F1: Actinomycin D structure and DNA interactions. (a) Chemical structure of ActinomycinD (ActD), with the planar phenoxazone ring system shown in green and pentapeptide side chains shown in red. (b) Ball and stick structure of two ActD molecules interacting with two DNA strands (different shades of blue) obtained from the pdb file IMNV, where phenoxazone rings (cyan for top molecule and green for bottom molecule) intercalate between DNA base pairs and the pentapeptide side chains (red) lie in the minor groove.
Mentions: Actinomycin D (ActD) is a DNA binding (1) small molecule with potent activity as an antibiotic (2) and anticancer agent (3). It is a neutral molecule that contains a planar tricyclic phenoxazone ring that intercalates dsDNA and two cyclic pentapeptide side chains (Figure 1a). ActD can intercalate between double stranded DNA (dsDNA) base pairs (4–8), bind to single-stranded DNA (ssDNA) (9–12) and can even ‘hemi-intercalate’ between the bases of a single DNA strand (13,14). Early studies found that once bound ActD dissociates slowly from dsDNA (4), with a component of its dissociation occurring on a time scale of ∼1000 s. These studies attributed ActD’s anticancer activity to this slow kinetics, and found it to be due to the slow fitting of its two highly stressed cyclic penta-peptide side chains into the DNA minor groove below and above the intercalated phenoxazone ring (4,15) (Figure 1b). The fitting into the groove is stabilized by hydrogen bonding of the ActD side chains to guanine bases (5–7), and associated with major DNA duplex deformations, such as strong bending (6,8), unwinding (6,16) and even base flipping (16,17). Duplex deformations are also driven by optimization of the tricyclic phenoxazone ring stacking with the 3′ faces of guanine (or adenine) residues in the opposite DNA strands (8,14,16). Competing models for the anticancer activity of ActD depend on the favored binding mode; Intercalation may inhibit replication by stabilizing dsDNA in front of the replication fork (8), while binding to destabilized duplexes such as transcription bubbles may inhibit DNA transcription (18–20), and ssDNA binding may directly stall the DNA polymerase (12). However, despite many years of study by a variety of methods and detailed knowledge of the relationship between DNA sequence, structure and the strength of ActD–DNA interactions, there is no consensus for any of these models and the reason for the selective anti-cancer activity of ActD at low concentrations remains unclear.Figure 1.

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