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

Thayaparan Paramanathan, Ioana Vladescu, Micah J. McCauley, Ioulia Rouzina, Mark C. Williams

Research output: Contribution to journalArticle

43 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)4925-4932
Number of pages8
JournalNucleic acids research
Volume40
Issue number11
DOIs
StatePublished - Jun 1 2012

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    Paramanathan, T., Vladescu, I., McCauley, M. J., Rouzina, I., & Williams, M. C. (2012). Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of Actinomycin D. Nucleic acids research, 40(11), 4925-4932. https://doi.org/10.1093/nar/gks069