TY - JOUR
T1 - Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of Actinomycin D
AU - Paramanathan, Thayaparan
AU - Vladescu, Ioana
AU - McCauley, Micah J.
AU - Rouzina, Ioulia
AU - Williams, Mark C.
N1 - Funding Information:
National Institutes of Health (GM-072462); the National Science Foundation (MCB-0744456). Funding for open access charge: National Institutes of Health (GM-072462).
PY - 2012/6
Y1 - 2012/6
N2 - 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 ActDDNA-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 (∼10nM), essentially locking ActD within dsDNA due to its slow dissociation, blocking RNA synthesis and leading to cell death.
AB - 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 ActDDNA-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 (∼10nM), essentially locking ActD within dsDNA due to its slow dissociation, blocking RNA synthesis and leading to cell death.
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U2 - 10.1093/nar/gks069
DO - 10.1093/nar/gks069
M3 - Article
C2 - 22328730
AN - SCOPUS:84862229609
VL - 40
SP - 4925
EP - 4932
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 11
ER -