A Holliday junction is a branched nucleic acid structure where four double-stranded domains are joined together. In biology, Holliday junctions are an intermediate in genetic recombination; these junctions usually have a symmetrical sequence and are thus mobile. Immobile Holliday junctions were artificially created by scientists at first to study their structure as a model for natural Holliday junctions, but they also later found use as basic structural building blocks in DNA nanotechnology. The structure is named after Robin Holliday, who proposed its existence in 1964.
Holliday junctions may exist in a number of different conformational isomers with different patterns of coaxial stacking between the four double-helical domains. Coaxial stacking is the tendency of nucleic acid blunt ends to bind to each other due to interactions between the exposed bases. There are three possible stacking conformers: an unstacked form and two stacked forms. The unstacked form dominates in the absence of divalent cations such as Mg2+ due to electrostatic repulsion between the negatively charged backbones of the strands, while 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 the identities of which pairs of the arms are stacked with each other, and 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 0.3−0.4 ms with no ions to 270−300 ms with 10 mM Mg2+. The change is 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 are known in some functional biological RNAs, such as U1 spliceosomal RNA and the hairpin ribozyme of the tobacco ringspot virus. 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.
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, 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 (DSBR) pathway (sometimes called the double Holliday junction model) and the synthesis-dependent strand annealing (SDSA) 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 here.
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 (if in same orientation during double strand break repair) or splice products (if in different orientations during double strand break repair). RuvA and RuvB are branch migration proteins, while RuvC is a junction-resolving enzyme.
There is also 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.
Use in DNA nanotechnology
DNA nanotechnology is the design and manufacture of artificial nucleic acid structures as engineering materials for nanotechnology rather than as the carriers of genetic information in living cells. The field uses branched DNA structures as fundamental components to create more complex, rationally designed structures. Holliday junctions are thus components of many such DNA structures. As isolated Holliday junction complexes are too flexible to assemble into large ordered arrays, structural motifs with multiple Holliday junctions are used to create rigid "tiles" that can then assemble into larger "arrays".
The most common such motif is the double crossover (DX) complex, which contains two Holliday junctions in close proximity to each other, resulting in a rigid structure that can self-assemble into larger arrays. The structure of the DX molecule forces the Holliday junctions to adopt a conformation with the double-helical domains directly side-by-side, in contrast to their preferred angle of about 60°. The complex can theoretically be designed to force the junctions into either a parallel or antiparallel orientation, but in practice the antiparallel variety are more well-behaved, and the parallel version is rarely used.
The DX structural motif is also the fundamental building block of the DNA origami method, which is used to make larger two- and three-dimensional structures of arbitrary shape. Instead of using individual DX tiles, a single long scaffold strand is folded into the desired shape by a number of short staple strands. When assembled, the scaffold strand is continuous through the double-helical domains, while the staple strands participate in the Holliday junctions as crossover strands.
A number of tile types have been demonstrated that retain the Holliday junction's native 60° angle. One such array uses tiles containing four Holliday junctions in a parallelogram arrangement. This structure had the benefit of allowing the junction angle to be directly visualized via atomic force microscopy. In addition, tiles of three Holliday junctions in a triangular fashion based on the principle of tensegrity has been used to make periodic three-dimensional arrays intended for use in X-ray crystallography of biomolecules.
Robin Holliday proposed the structure as part of his model of homologous recombination in 1964, based on his research on the organisms Ustilago maydis and Saccharomyces cerevisiae. The model provided a molecular mechanism that explained both gene conversion and chromosomal crossover. Notably, Holliday realized that the proposed pathway would create heteroduplex DNA segments with base mismatches between different versions of a single gene, and postulated that the cell must have a mechanism for mismatch repair, which was later discovered. Prior to Holliday's model, the accepted model involved the copy-choice mechanism.
In the original Holliday model for homologous recombination, single-strand breaks occur at the same point on one strand of each parental DNA. Free ends of each broken strand then migrate across to the other DNA helix, where the invading strands are joined to the free ends they encounter, resulting in the Holliday junction. As each crossover strand reanneals to its original partner strand it displaces the original complementary strand ahead of it, causing the Holliday junction to migrate, creating the heteroduplex segments. Depending on which strand was used as a template to repair the other, the four cells resulting from meiosis might end up with three copies of one allele and only one of the other, instead of the normal two of each, a property known as gene conversion.
Holliday's original model assumed that heteroduplex DNA would be presence on both chromosomes, but experimental data on yeast refuted this, leading to an updated model by Matt Meselson and Charley Radding in 1975, which introduced the idea of the branch migration. Further observations in the 1980s led to the proposal of alternate mechanisms for recombination such as the double-strand break model (by Jack Szostak, Frank Stahl, and others) and the single-strand annealing model. A third, the synthesis-dependent strand annealing model, did not involve Holliday junctions.
The first experimental evidence of the Holliday junction's structure came from electron microscopy studies in the late 1970s, where the four-arm structure was clearly visible in images of plasmid and bacteriophage DNA. Later in the 1980s, enzymes responsible for initiating the formation of, and binding to, Holliday junctions were identified, although as of 2004 the identification of mammalian Holliday junction resolvases remained elusive. In 1983, artificial Holliday junction molecules were first constructed from synthetic oligonucleotides by Nadrian Seeman, allowing for more direct study of their physical properties. Much of the early analysis of Holliday junction structure was inferred from gel electrophoresis, FRET, and hydroxyl radical and nuclease footprinting studies. In the 1990s, crystallography and nucleic acid NMR methods became available, as well as computational molecular modelling tools.
Initially, geneticists assumed that the junction would adopt a parallel rather than antiparallel conformation because that would place the homologous duplexes in closer alignment to each other. Chemical analysis in the 1980s showed that the junction actually preferred the antiparallel conformation, a finding that was considered controversial, with Robin Holliday himself initially doubting the findings. The antiparallel structure later became widely accepted due to X-ray crystallography data on in vitro molecules, although as of 2004 the implications for the in vivo structure remained unclear, especially the structure of the junctions is often altered by proteins bound to it.
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- Holliday junctions at the US National Library of Medicine Medical Subject Headings (MeSH)
- Conformational Change of Holliday Junction
- Analysis of branch migration activities of proteins using synthetic DNA substrates (a protocol)