Publication Date

5-2011

Advisor(s)

Ishita Mukerji

Department

Molecular Biology and Biochemistry

Language

English

Abstract

Holliday Junctions are critical DNA intermediates during the process of repairing double strand breaks in chromosomes and in homologous recombination. Junctions can occupy two forms, the open, X-shaped conformation, and the stacked, pseudo-duplex form. The transition between the two is normally mediated by proteins involved in recombination and repair; however, high ion concentration has been shown to induce stacked conformations. This goal of this study has been to understand the mechanism through which ions control junction conformation by gathering information about the junction’s ion-binding sites.

We have used the methodology of Förster Resonance Energy Transfer (FRET) between fluorophores on adjacent arms of the junction to investigate the conformation of the junction for the following ions: Mg2+, Ca2+, Eu3+, Tb3+, and [Co(NH3)6]3+. Titrations of ions, as monitored by FRET, have allowed us to measure the binding affinity and the degree to which each ion can fold the junction, suggesting that ion binding directly controls junction folding. This relationship of ion size and charge to degree of junction folding suggests that folding is regulated by ion binding to the core of the junction. Thermodynamic measurements of this transition yield along with van ‘t Hoff enthalpy and ITC experiments are in close precision.

An endothermic and exothermic reaction was detected, being attributed to ion binding and the resulting conformational change of the junction, and to the removal of water molecules from the inner and outer spheres of the ion. The junction-folding reaction contained an apparent stoichiometry of 1.86 +/- 0.187 sites. Lanthanide luminescence experiments have shown a homogeneity of ion-binding sites, a coordination by eight waters when junction bound out of nine maximum, and that these two ion-binding sites are within 6.6 angstrom of each other. These results are consistent with the binding to the central region of the junction, observed in several crystal structures and with MD simulations that predict binding to phosphates in two pockets of the junction’s core.

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