Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of Actinomycin D.
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.
Department of Physics, Northeastern University, Boston, MA-02115, USA.
Thayaparan
T
Paramanathan
Ioana
I
Vladescu
Micah J
MJ
McCauley
Ioulia
I
Rouzina
Mark C
MC
Williams
D000900:Anti-Bacterial Agents / Q000737:chemistry*; D000903:Antibiotics, Antineoplastic / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D003609:Dactinomycin / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D052898:Optical Tweezers; D013057:Spectrum Analysis / Q000379:methods
D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D003609:Dactinomycin; D004247:DNA