Inmolecular biology, the termdouble helix[1] refers to the structure formed bydouble-stranded molecules ofnucleic acids such asDNA. The doublehelical structure of a nucleic acid complex arises as a consequence of itssecondary structure, and is a fundamental component in determining itstertiary structure. The structure was discovered byMaurice Wilkins,Rosalind Franklin, her studentRaymond Gosling,James Watson, andFrancis Crick,[2] while the term "double helix" entered popular culture with the 1968 publication of Watson'sThe Double Helix: A Personal Account of the Discovery of the Structure of DNA.
The DNA double helixbiopolymer ofnucleic acid is held together bynucleotides whichbase pair together.[3] InB-DNA, the most common double helical structure found in nature, the double helix is right-handed with about 10–10.5 base pairs per turn.[4] The double helix structure of DNA contains amajor groove andminor groove. In B-DNA the major groove is wider than the minor groove.[3] Given the difference in widths of the major groove and minor groove, many proteins which bind to B-DNA do so through the wider major groove.[5]
The double-helix model ofDNA structure was first published in the journalNature byJames Watson andFrancis Crick in 1953,[6] (X,Y,Z coordinates in 1954[7]) based on the work ofRosalind Franklin and her studentRaymond Gosling, who took the crucial X-ray diffraction image of DNA labeled as "Photo 51",[8][9] andMaurice Wilkins,Alexander Stokes, andHerbert Wilson,[10] and base-pairing chemical and biochemical information byErwin Chargaff.[11][12][13][14][15][16] Before this,Linus Pauling—who had already accurately characterised the conformation of protein secondary structure motifs—and his collaboratorRobert Corey had posited, erroneously, that DNA would adopt atriple-stranded conformation.[17]
The realization that the structure of DNA is that of a double-helix elucidated the mechanism ofbase pairing by which genetic information is stored and copied in living organisms and is widely considered one of the most important scientific discoveries of the 20th century. Crick, Wilkins, and Watson each received one-third of the 1962Nobel Prize in Physiology or Medicine for their contributions to the discovery.[18]
Hybridization is the process ofcomplementarybase pairs binding to form a double helix. Melting is the process by which the interactions between the strands of the double helix are broken, separating the two nucleic acid strands. These bonds are weak, easily separated by gentle heating,enzymes, or mechanical force. Melting occurs preferentially at certain points in the nucleic acid.[19]T andA rich regions are more easily melted thanC andG rich regions. Some base steps (pairs) are also susceptible to DNA melting, such asT A andT G.[20] These mechanical features are reflected by the use of sequences such asTATA at the start of many genes to assist RNA polymerase in melting the DNA for transcription.
Strand separation by gentle heating, as used inpolymerase chain reaction (PCR), is simple, providing the molecules have fewer than about 10,000 base pairs (10 kilobase pairs, or 10 kbp). The intertwining of the DNA strands makes long segments difficult to separate.[21] The cell avoids this problem by allowing its DNA-melting enzymes (helicases) to work concurrently withtopoisomerases, which can chemically cleave the phosphate backbone of one of the strands so that it can swivel around the other.[22]Helicases unwind the strands to facilitate the advance of sequence-reading enzymes such asDNA polymerase.[23]
The geometry of a base, or base pair step can be characterized by 6 coordinates: shift, slide, rise, tilt, roll, and twist. These values precisely define the location and orientation in space of every base or base pair in a nucleic acid molecule relative to its predecessor along the axis of the helix. Together, they characterize the helical structure of the molecule. In regions of DNA or RNA where thenormal structure is disrupted, the change in these values can be used to describe such disruption.
For each base pair, considered relative to its predecessor, there are the following base pair geometries to consider:[24][25][26]
Rise and twist determine the handedness and pitch of the helix. The other coordinates, by contrast, can be zero. Slide and shift are typically small in B-DNA, but are substantial in A- and Z-DNA. Roll and tilt make successive base pairs less parallel, and are typically small.
"Tilt" has often been used differently in the scientific literature, referring to the deviation of the first, inter-strand base-pair axis from perpendicularity to the helix axis. This corresponds to slide between a succession of base pairs, and in helix-based coordinates is properly termed "inclination".
At least three DNA conformations are believed to be found in nature,A-DNA,B-DNA, andZ-DNA. TheB form described byJames Watson andFrancis Crick is believed to predominate in cells.[27] It is 23.7Å wide and extends 34 Å per 10bp of sequence. The double helix has a right-hand twist that makes one complete turn about its axis every 10.4–10.5 base pairs in solution. This frequency of twist (termed the helicalpitch) depends largely on stacking forces that each base exerts on its neighbours in the chain. Theabsolute configuration of the bases determines the direction of the helical curve for a given conformation.
A-DNA and Z-DNA differ significantly in their geometry and dimensions to B-DNA, although still form helical structures. It was long thought that the A form only occurs in dehydrated samples of DNA in the laboratory, such as those used incrystallographic experiments, and in hybrid pairings of DNA andRNA strands, but DNA dehydration does occurin vivo, andA-DNA is now known to have biological functions. Segments of DNA that cells havemethylated for regulatory purposes may adopt the Z geometry, in which the strands turn about the helical axis the opposite way to A-DNA and B-DNA. There is also evidence of protein-DNA complexes forming Z-DNA structures.
Other conformations are possible; A-DNA, B-DNA,C-DNA, E-DNA,[28]L-DNA (theenantiomeric form ofD-DNA),[29] P-DNA,[30] S-DNA, Z-DNA, etc. have been described so far.[31] In fact, only the letters F, Q, U, V, and Y are now[update] available to describe any new DNA structure that may appear in the future.[32][33] However, most of these forms have been created synthetically and have not been observed in naturally occurring biological systems.[citation needed] There are alsotriple-stranded DNA forms and quadruplex forms such as theG-quadruplex and thei-motif.
| Geometry attribute | A-DNA | B-DNA | Z-DNA |
|---|---|---|---|
| Helix sense | right-handed | right-handed | left-handed |
| Repeating unit | 1 bp | 1 bp | 2 bp |
| Rotation/bp | 32.7° | 34.3° | 60°/2 |
| bp/turn | 11 | 10.5 | 12 |
| Inclination of bp to axis | +19° | −1.2° | −9° |
| Rise/bp along axis | 2.3 Å (0.23 nm) | 3.32 Å (0.332 nm) | 3.8 Å (0.38 nm) |
| Pitch/turn of helix | 28.2 Å (2.82 nm) | 33.2 Å (3.32 nm) | 45.6 Å (4.56 nm) |
| Mean propeller twist | +18° | +16° | 0° |
| Glycosyl angle | anti | anti | C: anti, G: syn |
| Sugar pucker | C3'-endo | C2'-endo | C: C2'-endo, G: C2'-exo |
| Diameter | 23 Å (2.3 nm) | 20 Å (2.0 nm) | 18 Å (1.8 nm) |
Twin helical strands form the DNA backbone. Another double helix may be found by tracing the spaces, or grooves, between the strands. These voids are adjacent to the base pairs and may provide abinding site.[37] As the strands are not directly opposite each other, the grooves are unequally sized. One groove, the major groove, is 22 Å wide and the other, the minor groove, is 12 Å wide.[38] The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. As a result, proteins liketranscription factors that can bind to specific sequences in double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove.[5] This situation varies in unusual conformations of DNA within the cell(see below), but the major and minor grooves are always named to reflect the differences in size that would be seen if the DNA is twisted back into the ordinary B form.[39]
Alternativenon-helical models were briefly considered in the late 1970s as a potential solution to problems inDNA replication inplasmids andchromatin. However, the models were set aside in favor of the double-helical model due to subsequent experimental advances such asX-ray crystallography of DNA duplexes and later thenucleosome core particle, and the discovery oftopoisomerases. Also, the non-double-helical models are not currently accepted by the mainstream scientific community.[40][41]
DNA is a relatively rigid polymer, typically modelled as aworm-like chain. It has three significant degrees of freedom; bending, twisting, and compression, each of which cause certain limits on what is possible with DNA within a cell. Twisting-torsional stiffness is important for the circularisation of DNA and the orientation of DNA bound proteins relative to each other and bending-axial stiffness is important for DNA wrapping and circularisation and protein interactions. Compression-extension is relatively unimportant in the absence of high tension.
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| Sequence | Persistence length / base pairs |
|---|---|
| Random | 154±10 |
| (CA)repeat | 133±10 |
| (CAG)repeat | 124±10 |
| (TATA)repeat | 137±10 |
DNA in solution does not take a rigid structure but is continually changing conformation due to thermal vibration and collisions with water molecules, which makes classical measures of rigidity impossible to apply. Hence, the bending stiffness of DNA is measured by the persistence length, defined as:
Bending flexibility of a polymer is conventionally quantified in terms of its persistence length, Lp, a length scale below which the polymer behaves more or less like a rigid rod. Specifically, Lp is defined as length of the polymer segment over which the time-averaged orientation of the polymer becomes uncorrelated...[42]
This value may be directly measured using anatomic force microscope to directly image DNA molecules of various lengths. In an aqueous solution, the average persistence length has been found to be of around 50 nm (or 150 base pairs).[43] More broadly, it has been observed to be between 45 and 60 nm[44] or 132–176 base pairs (the diameter of DNA is 2 nm)[45] This can vary significantly due to variations in temperature, aqueous solution conditions and DNA length.[44] This makes DNA a moderately stiff molecule.[43]
The persistence length of a section of DNA is somewhat dependent on its sequence, and this can cause significant variation. The variation is largely due to base stacking energies and the residues which extend into theminor andmajor grooves.
| Step | Stacking ΔG /kcal mol−1 |
|---|---|
| T A | -0.19 |
| T G orC A | -0.55 |
| C G | -0.91 |
| A G orC T | -1.06 |
| A A orT T | -1.11 |
| A T | -1.34 |
| G A orT C | -1.43 |
| C C orG G | -1.44 |
| A C orG T | -1.81 |
| G C | -2.17 |
At length-scales larger than thepersistence length, the entropic flexibility of DNA is remarkably consistent with standardpolymer physics models, such as theKratky-Porodworm-like chain model.[47] Consistent with theworm-like chain model is the observation that bending DNA is also described byHooke's law at very small (sub-piconewton) forces. For DNA segments less than the persistence length, the bending force is approximately constant and behaviour deviates from the worm-like chain predictions.
This effect results in unusual ease in circularising small DNA molecules and a higher probability of finding highly bent sections of DNA.[48]
DNA molecules often have a preferred direction to bend, i.e.,anisotropic bending. This is, again, due to the properties of the bases which make up the DNA sequence - a random sequence will have no preferred bend direction, i.e., isotropic bending.
Preferred DNA bend direction is determined by the stability of stacking each base on top of the next. If unstable base stacking steps are always found on one side of the DNA helix then the DNA will preferentially bend away from that direction. As bend angle increases then steric hindrances and ability to roll the residues relative to each other also play a role, especially in the minor groove.A andT residues will be preferentially be found in the minor grooves on the inside of bends. This effect is particularly seen in DNA-protein binding where tight DNA bending is induced, such as innucleosome particles. See base step distortions above.
DNA molecules with exceptional bending preference can become intrinsically bent. This was first observed intrypanosomatidkinetoplast DNA. Typical sequences which cause this contain stretches of 4-6T andA residues separated byG andC rich sections which keep the A and T residues in phase with the minor groove on one side of the molecule. For example:
| ¦ | ¦ | ¦ | ¦ | ¦ | ¦ | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| G | A | T | T | C | C | C | A | A | A | A | A | T | G | T | C | A | A | A | A | A | A | T | A | G | G | C | A | A | A | A | A | A | T | G | C | C | A | A | A | A | A | A | T | C | C | C | A | A | A | C |
The intrinsically bent structure is induced by the 'propeller twist' of base pairs relative to each other allowing unusual bifurcated Hydrogen-bonds between base steps. At higher temperatures this structure is denatured, and so the intrinsic bend is lost.
All DNA which bends anisotropically has, on average, a longer persistence length and greater axial stiffness. This increased rigidity is required to prevent random bending which would make the molecule act isotropically.
DNA circularization depends on both the axial (bending) stiffness and torsional (rotational) stiffness of the molecule. For a DNA molecule to successfully circularize it must be long enough to easily bend into the full circle and must have the correct number of bases so the ends are in the correct rotation to allow bonding to occur. The optimum length for circularization of DNA is around 400 base pairs (136 nm)[citation needed], with an integral number of turns of the DNA helix, i.e., multiples of 10.4 base pairs. Having a non integral number of turns presents a significantenergy barrier for circularization, for example a 10.4 x 30 = 312 base pair molecule will circularize hundreds of times faster than 10.4 x 30.5 ≈ 317 base pair molecule.[49]
The bending of short circularized DNA segments is non-uniform. Rather, for circularized DNA segments less than the persistence length, DNA bending is localised to 1-2 kinks that form preferentially in AT-rich segments. If anick is present, bending will be localised to the nick site.[48]
Longer stretches of DNA are entropically elastic under tension. When DNA is in solution, it undergoes continuous structural variations due to the energy available in thethermal bath of the solvent. This is due to the thermal vibration of the molecule combined with continual collisions with water molecules. Forentropic reasons, more compact relaxed states are thermally accessible than stretched out states, and so DNA molecules are almost universally found in a tangled relaxed layouts. For this reason, one molecule of DNA will stretch under a force, straightening it out. Usingoptical tweezers, the entropic stretching behavior of DNA has been studied and analyzed from apolymer physics perspective, and it has been found that DNA behaves largely like theKratky-Porodworm-like chain model under physiologically accessible energy scales.
Under sufficient tension and positive torque, DNA is thought to undergo aphase transition with the bases splaying outwards and the phosphates moving to the middle. This proposed structure for overstretched DNA has been calledP-form DNA, in honor ofLinus Pauling who originally presented it as a possible structure of DNA.[30]
Evidence from mechanical stretching of DNA in the absence of imposed torque points to a transition or transitions leading to further structures which are generally referred to asS-form DNA. These structures have not yet been definitively characterised due to the difficulty of carrying out atomic-resolution imaging in solution while under applied force although many computer simulation studies have been made (for example,[50][51]).
Proposed S-DNA structures include those which preserve base-pair stacking and hydrogen bonding (GC-rich), while releasing extension by tilting, as well as structures in which partial melting of the base-stack takes place, while base-base association is nonetheless overall preserved (AT-rich).
Periodic fracture of the base-pair stack with a break occurring once per three bp (therefore one out of every three bp-bp steps) has been proposed as a regular structure which preserves planarity of the base-stacking and releases the appropriate amount of extension,[52] with the term "Σ-DNA" introduced as a mnemonic, with the three right-facing points of the Sigma character serving as a reminder of the three grouped base pairs. The Σ form has been shown to have a sequence preference for GNC motifs which are believed under theGNC hypothesis to be of evolutionary importance.[53]
The B form of the DNA helix twists 360° per 10.4-10.5 bp in the absence of torsional strain. But many molecular biological processes can induce torsional strain. A DNA segment with excess or insufficient helical twisting is referred to, respectively, as positively or negativelysupercoiled. DNAin vivo is typically negatively supercoiled, which facilitates the unwinding (melting) of the double-helix required for RNAtranscription.
Within the cell most DNA is topologically restricted. DNA is typically found in closed loops (such asplasmids in prokaryotes) which are topologically closed, or as very long molecules whose diffusion coefficients produce effectively topologically closed domains. Linear sections of DNA are also commonly bound to proteins or physical structures (such as membranes) to form closed topological loops.
Francis Crick was one of the first to propose the importance of linking numbers when considering DNA supercoils. In a paper published in 1976, Crick outlined the problem as follows:
In considering supercoils formed by closed double-stranded molecules of DNA certain mathematical concepts, such as the linking number and the twist, are needed. The meaning of these for a closed ribbon is explained and also that of the writhing number of a closed curve. Some simple examples are given, some of which may be relevant to the structure of chromatin.[54]
Analysis of DNA topology uses three values:
Any change of T in a closed topological domain must be balanced by a change in W, and vice versa. This results in higher order structure of DNA. A circular DNA molecule with a writhe of 0 will be circular. If the twist of this molecule is subsequently increased or decreased by supercoiling then the writhe will be appropriately altered, making the molecule undergo plectonemic or toroidal superhelical coiling.
When the ends of a piece of double stranded helical DNA are joined so that it forms a circle the strands aretopologically knotted. This means the single strands cannot be separated any process that does not involve breaking a strand (such as heating). The task of un-knotting topologically linked strands of DNA falls to enzymes termedtopoisomerases. These enzymes are dedicated to un-knotting circular DNA by cleaving one or both strands so that another double or single stranded segment can pass through. This un-knotting is required for the replication of circular DNA and various types ofrecombination in linear DNA which have similar topological constraints.
For many years, the origin of residual supercoiling in eukaryotic genomes remained unclear. This topological puzzle was referred to by some as the "linking number paradox".[55] However, when experimentally determined structures of thenucleosome displayed an over-twisted left-handed wrap of DNA around thehistone octamer,[56][57] thisparadox was considered to be solved by the scientific community.
However, the discovery of topoisomerases took "the sting" out of the topological objection to the plectonaemic double helix. The more recent solution of the single crystal X-ray structure of the nucleosome core particle showed nearly 150 base pairs of the DNA (i.e., about 15 complete turns), with a structure that is in all essential respects the same as the Watson–Crick model. This dealt a death blow to the idea that other forms of DNA, particularly double helical DNA, exist as anything other than local or transient structures.[permanent dead link]