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

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 thermo...

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Main Authors: Jacob L. Litke, Yan Li, Laura M. Nocka, Ishita Mukerji
Format: Article
Language:English
Published: MDPI AG 2016-03-01
Series:International Journal of Molecular Sciences
Subjects:
Online Access:http://www.mdpi.com/1422-0067/17/3/366
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spelling doaj-b1b52ba67ef047d59edc6496583f05692020-11-24T21:10:33ZengMDPI AGInternational Journal of Molecular Sciences1422-00672016-03-0117336610.3390/ijms17030366ijms17030366Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)Jacob L. Litke0Yan Li1Laura M. Nocka2Ishita Mukerji3Department of Molecular Biology and Biochemistry and Molecular Biophysics Program, Wesleyan University, Middletown, CT 06459-0175, USADepartment of Molecular Biology and Biochemistry and Molecular Biophysics Program, Wesleyan University, Middletown, CT 06459-0175, USADepartment of Molecular Biology and Biochemistry and Molecular Biophysics Program, Wesleyan University, Middletown, CT 06459-0175, USADepartment of Molecular Biology and Biochemistry and Molecular Biophysics Program, Wesleyan University, Middletown, CT 06459-0175, USAHolliday 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.http://www.mdpi.com/1422-0067/17/3/366Holliday junctionsnucleic acidsFRETion-bindinglanthanide luminescence
collection DOAJ
language English
format Article
sources DOAJ
author Jacob L. Litke
Yan Li
Laura M. Nocka
Ishita Mukerji
spellingShingle Jacob L. Litke
Yan Li
Laura M. Nocka
Ishita Mukerji
Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)
International Journal of Molecular Sciences
Holliday junctions
nucleic acids
FRET
ion-binding
lanthanide luminescence
author_facet Jacob L. Litke
Yan Li
Laura M. Nocka
Ishita Mukerji
author_sort Jacob L. Litke
title Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)
title_short Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)
title_full Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)
title_fullStr Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)
title_full_unstemmed Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)
title_sort probing the ion binding site in a dna holliday junction using förster resonance energy transfer (fret)
publisher MDPI AG
series International Journal of Molecular Sciences
issn 1422-0067
publishDate 2016-03-01
description 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.
topic Holliday junctions
nucleic acids
FRET
ion-binding
lanthanide luminescence
url http://www.mdpi.com/1422-0067/17/3/366
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AT yanli probingtheionbindingsiteinadnahollidayjunctionusingforsterresonanceenergytransferfret
AT lauramnocka probingtheionbindingsiteinadnahollidayjunctionusingforsterresonanceenergytransferfret
AT ishitamukerji probingtheionbindingsiteinadnahollidayjunctionusingforsterresonanceenergytransferfret
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