The Mechanism of DNA Junction Melting

Four-way Holliday junctions play vital roles in genetic recombination, DNA damage repair, and other mechanisms of chromosomal rearrangement. Here we examine the melting of Holliday junctions through a combination of FRET experiments and MD simulations. We show that melting of this junction initiates at specific locations. At physiological salt concentrations, the junction “folds” into a stacked conformation consisting of two crossing pseudo-duplexes, and our results indicate that melting proceeds as a quasi-independent process along these pseudo-duplexes, rather than as a uniform process throughout the junction. We interrogate the melting locally by measuring FRET from fluorescent nucleotide analogs strategically placed at several locations in the junction. To help interpret the FRET data, we use the coarse-grained 3SPN.2 model to simulate the junction melting. By analyzing an ensemble of simulations, we describe approaches to characterize junction conformation and the dynamics of melting. Our results demonstrate the ability of the 3SPN.2 model to predict structural and dynamic aspects of Holliday junctions and offer a two-step mechanism for junction melting.