Investigation of the Melting Thermodynamics of a DNA 4-Way Junction: One Base at a Time

Abstract

DNA four-way junctions are branched structures that form between two homologous chromosomes. These junctions play key roles during several cellular processes, including meiosis and DNA double-stranded break repair, however their mechanism of formation and separation are not well understood, particularly with respect to branch migration. We have investigated the thermodynamic stability of the DNA four-way junction J3 using the fluorescent pteridine nucleoside analogues, 6-MAP and 6-MI, which provide site-specific information on the melting process. We have incorporated these probes at different locations throughout the junction to determine the influence of position on junction stability. Preliminary fluorescence data suggest that the central region of the four-way junction, a region under much torsional strain, is in fact more stable than previously hypothesized. We have also investigated the relative stability of the different arms, by incorporating probes on each arm approximately the same distance from the junction center. These results are compared with those predicted from coarse-grained simulations of the junction using the 3 sites per nucleotide (3SPN.2) model. Already, we have demonstrated the ability of this model to reproduce many experimentally determined aspects of DNA junction structure and stability, including the temperature dependence of melting on salt concentration, the bias between open and stacked conformations, the relative populations of conformers at high salt concentration, and the inter-duplex angle between arms. We are now using a replica-exchange molecular dynamics approach to evaluate the fraction of bonded bases along each arm of the junction over the temperature range associated with melting. This approach allows us to determine base-by-base the local melting temperature along the arm. We specifically compare the patterns of melting observed experimentally with those obtained from the coarse-grained simulations.