Next Article in Journal
Geranylgeraniol and Neurological Impairment: Involvement of Apoptosis and Mitochondrial Morphology
Previous Article in Journal
Spinal Glutamate Transporters Are Involved in the Development of Electroacupuncture Tolerance
Previous Article in Special Issue
Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics
Article Menu
Issue 3 (March) cover image

Export Article

Open AccessArticle
Int. J. Mol. Sci. 2016, 17(3), 366; doi:10.3390/ijms17030366

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)
View Full-Text   |   Download PDF [2963 KB, uploaded 11 March 2016]   |  

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. View Full-Text
Keywords: Holliday junctions; nucleic acids; FRET; ion-binding; lanthanide luminescence Holliday junctions; nucleic acids; FRET; ion-binding; lanthanide luminescence
Figures

Figure 1

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

Supplementary material

Scifeed alert for new publications

Never miss any articles matching your research from any publisher
  • Get alerts for new papers matching your research
  • Find out the new papers from selected authors
  • Updated daily for 49'000+ journals and 6000+ publishers
  • Define your Scifeed now

SciFeed Share & Cite This Article

MDPI and ACS Style

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.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top