A Holliday junction is a branched nucleic acid structure that contains four double-stranded arms joined. These arms may adopt one of several conformations depending on buffer salt concentrations and the sequence of nucleobases closest to the junction. The structure is named after Robin Holliday, the molecular biologist who proposed its existence in 1964.

In biology, Holliday junctions are a key intermediate in many types of genetic recombination, as well as in double-strand break repair. These junctions usually have a symmetrical sequence and are thus mobile, meaning that the four individual arms may slide through the junction in a specific pattern that largely preserves base pairing. Additionally, four-arm junctions similar to Holliday junctions appear in some functional RNA molecules.

Immobile Holliday junctions, with asymmetrical sequences that lock the strands in a specific position, were artificially created by scientists to study their structure as a model for natural Holliday junctions. These junctions also later found use as basic structural building blocks in DNA nanotechnology, where multiple Holliday junctions can be combined into specific designed geometries that provide molecules with a high degree of structural rigidity.

Holliday junctions may exist in a variety of conformational isomers with different patterns of coaxial stacking between the four double-helical arms. Coaxial stacking is the tendency of nucleic acid blunt ends to bind to each other, by interactions between the exposed bases. There are three possible conformers: an unstacked (or open-X) form and two stacked forms. The unstacked form dominates in the absence of divalent cations such as Mg²⁺, because of electrostatic repulsion between the negatively charged backbones of the strands. In the presence of at least about 0.1 mM Mg²⁺, the electrostatic repulsion is counteracted and the stacked structures predominate. As of 2000, it was not known with certainty whether the electrostatic shielding was the result of site-specific binding of cations to the junction, or the presence of a diffuse collection of the ions in solution.

The unstacked form is a nearly square planar, extended conformation. On the other hand, the stacked conformers have two continuous double-helical domains separated by an angle of about 60° in a right-handed direction. Two of the four strands stay roughly helical, remaining within each of the two double-helical domains, while the other two cross between the two domains in an antiparallel fashion.

The two possible stacked forms differ in which pairs of the arms are stacked with each other; which of the two dominates is highly dependent on the base sequences nearest to the junction. Some sequences result in an equilibrium between the two conformers, while others strongly prefer a single conformer. In particular, junctions containing the sequence A-CC bridging the junction point appear to strongly prefer the conformer that allows a hydrogen bond to form between the second cytosine and one of the phosphates at the junction point. While most studies have focused on the identities of the four bases nearest to the junction on each arm, it is evident that bases farther out can also affect the observed stacking conformations.

In junctions with symmetrical sequences, the branchpoint is mobile and can migrate in a random walk process. The rate of branch migration varies dramatically with ion concentration, with single-step times increasing from 0.3 to 0.4 ms with no ions to 270−300 ms with 10 mM Mg²⁺. The change in rate is correlated with the formation of the stacked versus the unstacked structures.

Holliday junctions with a nick, or break in one of the strands, at the junction point adopt a perpendicular orientation, and always prefer the stacking conformer that places the nick on a crossover strand rather than a helical strand.