Melting Behavior of a DNA Four-Way Junction Using Spectroscopic and Calorimetric Techniques

Abstract

Intramolecular four-way junctions are structures present during homologous recombination, repair of double stranded DNA breaks, and integron recombination. Because of the wide range of biological processes four-way junctions are involved in, understanding how and under what conditions these structures form is critical. In this work, we used a combination of spectroscopic and calorimetric techniques to present a complete thermodynamic description of the unfolding of a DNA four-way junction (FWJ) and its appropriate control stem-loop motifs (Dumbbell, GAAATT-Hp, CTATC-Hp, GTGC-Hp, and GCGC-Hp). The overall results show that the four-way junction increases the cooperative unfolding of its stems, although the reason for this is unclear, as the arms do not unfold as coaxial stacks, and thus its melting behavior cannot be accurately described by its control molecules. This is in contrast to what has been seen for two- and three-way junctions. In addition, the lack of base stacking and the ΔH/ΔH ratio seen at low salt indicate the four-way junction exists as a mixture of conformations, one of which is most likely the open-X structure which has unpaired bases at the junction. This was confirmed by single value decomposition of CD and UV spectra. This indicates that at low salt there is a third spectroscopically distinct species, while at higher salt there are only two species, folded and unfolded. Based on the enthalpy, Δn, and Δn, the dominant folded structure at high salt is most likely the antiparallel stacked-X structure.

A Thermodynamic Perspective on Potential G‐Quadruplex Structures as Silencer Elements in the MYC Promoter

“…From at hermodynamic perspective, an elevated potassium ion concentrationresultsinamore exothermicf olding of the parallel quadruplex, yeti sa ccompanied by ah igher entropic penalty.T his pointst os tronger intramolecular interactions and ah igher rigidity similart oo bservations reported for aD NA four-wayj unction lacking more specific cation binding. [26] Because the DH8 contribution to the Gibbs free energy predominates even at highert emperatures, the overall thermodynamic stability of the quadruplexes will always increase in the accessible temperature range at higher K + concentrations. On the other hand, relative stabilities amongq uadruplexes are anticipatedt on ot be affected by changing K + concentrations.…”

Quadruplex–Duplex Junction: A High‐Affinity Binding Site for Indoloquinoline Ligands

“…[11][12][13][14] DNA junctionsa re important elements in cellular maintenance processes. [15][16][17] Given that G-quadruplexf orming sequences in gene promoter regions originate from duplex DNA, [18] the presence of quadruplex-duplex (Q-D) motifs seems obvious. Q-D hybrid structures have already been re-ported2 5y ear ago [19] and severalv ariants have since been developed by placing the duplex forming sequence at different internal or externalp ositions of the G4.…”

“…DNA junctions are important elements in cellular maintenance processes [15–17] . Given that G‐quadruplex forming sequences in gene promoter regions originate from duplex DNA, [18] the presence of quadruplex–duplex (Q–D) motifs seems obvious.…”

Conformation and protein interactions of intramolecular DNA and phosphorothioate four-way junctions

“…18 More recently, Carr et al designed a thermostable intramolecular 4WJ that contains hairpins composed of five consecutive thymine residues. 19 We hypothesize that the mini-hairpins will also enhance resistance against exonucleases at least in part by effectively removing binding sites (e.g. free 3 0 and 5'-ends).…”