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Int. J. Mol. Sci. 2016, 17(3), 366;

Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)

Department of Molecular Biology and Biochemistry and Molecular Biophysics Program, Wesleyan University, Middletown, CT 06459-0175, USA
Author to whom correspondence should be addressed.
Academic Editor: Herbert Schneckenburger
Received: 6 November 2015 / Revised: 21 February 2016 / Accepted: 26 February 2016 / Published: 10 March 2016
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Holliday Junctions are critical DNA intermediates central to double strand break repair and homologous recombination. The junctions can adopt two general forms: open and stacked-X, which are induced by protein or ion binding. In this work, fluorescence spectroscopy, metal ion luminescence and thermodynamic measurements are used to elucidate the ion binding site and the mechanism of junction conformational change. Förster resonance energy transfer measurements of end-labeled junctions monitored junction conformation and ion binding affinity, and reported higher affinities for multi-valent ions. Thermodynamic measurements provided evidence for two classes of binding sites. The higher affinity ion-binding interaction is an enthalpy driven process with an apparent stoichiometry of 2.1 ± 0.2. As revealed by Eu3+ luminescence, this binding class is homogeneous, and results in slight dehydration of the ion with one direct coordination site to the junction. Luminescence resonance energy transfer experiments confirmed the presence of two ions and indicated they are 6–7 Å apart. These findings are in good agreement with previous molecular dynamics simulations, which identified two symmetrical regions of high ion density in the center of stacked junctions. These results support a model in which site-specific binding of two ions in close proximity is required for folding of DNA Holliday junctions into the stacked-X conformation. View Full-Text
Keywords: Holliday junctions; nucleic acids; FRET; ion-binding; lanthanide luminescence Holliday junctions; nucleic acids; FRET; ion-binding; lanthanide luminescence

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Litke, J.L.; Li, Y.; Nocka, L.M.; Mukerji, I. Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET). Int. J. Mol. Sci. 2016, 17, 366.

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