Abstract
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.
Highlights
Holliday Junctions are composed of four DNA strands and are central to the process of homologous recombination
Europium excitation spectra and luminescence lifetime measurements were made using a spectroscopic system powered by an Nd:YAG laser coupled with a master oscillator power oscillator (MOPO) (Department of Chemistry, University at Buffalo, State University of New York, Buffalo, New York)
We have used a number of different methods to examine the ion binding sites in a DNA 4WJ. These results collectively support a model in which ion binding to the junction is largely mediated by electrostatic interactions, where multivalent ions bind with higher affinity than monovalent ions
Summary
Holliday Junctions are composed of four DNA strands and are central to the process of homologous recombination. We have assumed that the population of the high FRET conformer is 77% based upon populations determined in the gel and by fluorescence using single molecule and equilibrium energy transfer methods [14,17,18] These previous measurements were done with a number of different ion types at different concentrations and did not observe any changes in relative population. The current FRET measurements have further suggested that ion size potentially influences the amount of ion-induced conformational change This dependence of transfer efficiency on ion type is consistent with a model where ion binding is occurring at a specific site(s) within the junction and extends earlier findings [14]. Jl.yMcohl. aScrig. 2e0d16,io17n, s36a6re needed to aid in the neutralization of the phosphate backbone an dof r15educe repulcsrieoantin[1g0,a1n9–e2l1e]c.tronegative cleft, and the positively charged ions are needed to aid in the neutralization of the phosphate backbone and reduce repulsion [10,19,20,21]
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