Holliday junction

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.

Structure

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 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.
RNA Holliday junctions assume an antiparallel stacked conformation at high magnesium concentrations, a perpendicular stacked conformation at moderate concentrations, and rotate into a parallel stacked conformation at low concentrations, while even small calcium ion concentrations favor the antiparallel conformer.

Biological function

The Holliday junction is a key intermediate in homologous recombination, a biological process that increases genetic diversity by shifting genes between two chromosomes, as well as site-specific recombination events involving integrases. They are additionally involved in repair of double-strand breaks. In addition, cruciform structures involving Holliday junctions can arise to relieve helical strain in symmetrical sequences in DNA supercoils. While four-arm junctions also appear in functional RNA molecules, such as U1 spliceosomal RNA and the hairpin ribozyme of the tobacco ringspot virus, these usually contain unpaired nucleotides in between the paired double-helical domains, and thus do not strictly adopt the Holliday structure.
The Holliday junctions in homologous recombination are between identical or nearly identical sequences, leading to a symmetric arrangement of sequences around the central junction. This allows a branch migration process to occur where the strands move through the junction point. Cleavage, or resolution, of the Holliday junction can occur in two ways. Cleavage of the original set of strands leads to two molecules that may show gene conversion but not chromosomal crossover, while cleavage of the other set of two strands causes the resulting recombinant molecules to show crossover. All products, regardless of cleavage, are heteroduplexes in the region of Holliday junction migration.
Many proteins are able to recognize or distort the Holliday junction structure. One such class contains junction-resolving enzymes that cleave the junctions, sometimes in a sequence-specific fashion. Such proteins distort the structure of the junction in various ways, often pulling the junction into an unstacked conformation, breaking the central base pairs, and/or changing the angles between the four arms. Other classes are branch migration proteins that increase the exchange rate by orders of magnitude, and site-specific recombinases. In prokaryotes, Holliday junction resolvases fall into two families, integrases and nucleases, that are each structurally similar although their sequences are not conserved.
In eukaryotes, two primary models for how homologous recombination repairs double-strand breaks in DNA are the double-strand break repair pathway and the synthesis-dependent strand annealing pathway. In the case of double strand breakage, the 3' end is degraded and the longer 5' end invades the contiguous sister chromatid, forming a replication bubble. As this bubble nears the broken DNA, the longer 5' antisense strand again invades the sense strand of this portion of DNA, transcribing a second copy. When replication ends, both tails are reconnected to form two Holliday Junctions, which are then cleaved in a variety of patterns by proteins. An animation of this process can be seen .
Double-strand DNA breaks in bacteria are repaired by the RecBCD pathway of homologous recombination. Breaks that occur on only one of the two DNA strands, known as single-strand gaps, are thought to be repaired by the RecF pathway. Both the RecBCD and RecF pathways include a series of reactions known as branch migration, in which single DNA strands are exchanged between two intercrossed molecules of duplex DNA, and resolution, in which those two intercrossed molecules of DNA are cut apart and restored to their normal double-stranded state. Homologous recombination occurs in several groups of viruses. In DNA viruses such as herpesvirus, recombination occurs through a break-and-rejoin mechanism like in bacteria and eukaryotes. In bacteria, branch migration is facilitated by the RuvABC complex or RecG protein, molecular motors that use the energy of ATP hydrolysis to move the junction. The junction must then be resolved into two separate duplexes, restoring either the parental configuration or a crossed-over configuration. Resolution can occur in either a horizontal or vertical fashion during homologous recombination, giving patch products or splice products. RuvA and RuvB are branch migration proteins, while RuvC is a junction-resolving enzyme.
There is evidence for recombination in some RNA viruses, specifically positive-sense ssRNA viruses like retroviruses, picornaviruses, and coronaviruses. There is controversy over whether homologous recombination occurs in negative-sense ssRNA viruses like influenza.

Resolution

In budding yeast Saccharomyces cerevisiae, Holliday junctions can be resolved by four different pathways that account for essentially all Holliday junction resolution in vivo. The pathway that produces the majority of crossovers in S. cerevisiae budding yeast, and possibly in mammals, involves proteins EXO1, MLH1-MLH3 heterodimer and SGS1. The MLH1-MLH3 heterodimer binds preferentially to Holliday junctions. It is an endonuclease that makes single-strand breaks in supercoiled double-stranded DNA. The MLH1-MLH3 heterodimer promotes the formation of crossover recombinants. While the other three pathways, involving proteins MUS81-MMS4, SLX1 and YEN1, respectively, can promote Holliday junction resolution in vivo, absence of all three nucleases has only a modest impact on formation of crossover products.
Double mutants deleted for both MLH3 and MMS4 showed dramatically reduced crossing over compared to wild-type ; however spore viability was reasonably high and chromosomal disjunction appeared mostly functional.
Although MUS81 is a component of a minor crossover pathway in the meiosis of budding yeast, plants and vertebrates, in the protozoan Tetrahymena thermophila, MUS81 appears to be part of an essential, if not the predominant crossover pathway. The MUS81 pathway also appears to be the predominant crossover pathway in the fission yeast Schizosaccharomyces pombe.
The MSH4 and MSH5 proteins form a hetero-oligomeric structure in yeast and humans. In the yeast Saccharomyces cerevisiae MSH4 and MSH5 act specifically to facilitate crossovers between homologous chromosomes during meiosis. The MSH4/MSH5 complex binds and stabilizes double Holliday junctions and promotes their resolution into crossover products. An MSH4 hypomorphic mutant of S. cerevisiae showed a 30% genome wide reduction in crossover numbers, and a large number of meioses with non exchange chromosomes. Nevertheless, this mutant gave rise to spore viability patterns suggesting that segregation of non-exchange chromosomes occurred efficiently. Thus in S. cerevisiae proper segregation apparently does not entirely depend on crossovers between homologous pairs.
Recently, it was shown that CRISPR-associated Cas12a nuclease from the Acidominococcus bacteria can resolve Holliday junction in-vitro.