Parallel DNA Holliday junctions: Myth or reality? An atomistic molecular dynamics study.

Abstract

Holliday junctions (HJs) are key intermediates of homologous recombination and fundamental building blocks in DNA nanotechnology. Although the antiparallel stacked-X conformation has been extensively characterized, whether the parallel HJ conformations exist in aqueous solution remains unresolved. Here, we address this question using extensive atomistic molecular dynamics (MD) simulations and replica-exchange umbrella sampling (REUS) free-energy calculations. Starting from canonical antiparallel HJs, standard MD simulations occasionally revealed spontaneous transitions to parallel conformations on the microsecond timescale without disrupting the DNA duplexes or passing through an open junction intermediate. REUS free-energy profiles confirmed the antiparallel conformation as the global minimum while revealing a well-defined local minimum corresponding to the parallel conformation, thereby establishing the parallel conformation as thermodynamically metastable in solution. The combination of relatively low occupancy, microsecond-scale interconversion, and the surprisingly close structural similarity between antiparallel and parallel junctions provides a plausible explanation for the lack of direct experimental detection. These results reconcile the apparent absence of experimental evidence for parallel HJs with their structural feasibility in solution and offer a fresh perspective on the historical debate. The existence of metastable parallel HJs in solution may be relevant to supercoiled DNA duplexes and to nanotechnology, where such junctions enable novel lattice architectures.

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