Simplified representation of a double stranded DNA helix with coloured bases
Two complementary regions of nucleic acid molecules will bind and form a double helical structure held together by base pairs.
In molecular biology, the term double helix refers to the structure formed by double-stranded molecules of nucleic acids such as DNA. The double helical structure of a nucleic acid complex arises as a consequence of its secondary structure, and is a fundamental component in determining its tertiary structure. The term entered popular culture with the publication in 1968 of The Double Helix: A Personal Account of the Discovery of the Structure of DNA by James Watson.
The DNA double helix biopolymer of nucleic acid, held together by nucleotides which base pair together. In B-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. The double helix structure of DNA contains a major groove and minor groove. In B-DNA the major groove is wider than the minor groove.
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.
History
The double-helix model of DNA structure was first published in the journal Nature by James Watson and Francis Crick in 1953, (X,Y,Z coordinates in 1954) based upon the crucial X-ray diffraction image of DNA labeled as "Photo 51", from Rosalind Franklin in 1952, followed by her more clarified DNA image with Raymond Gosling, Maurice Wilkins, Alexander Stokes, and Herbert Wilson, and base-pairing chemical and biochemical information by Erwin Chargaff. The prior model was triple-stranded DNA.
The realization that the structure of DNA is that of a double-helix elucidated the mechanism of base 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 1962 Nobel Prize in Physiology or Medicine for their contributions to the discovery.
(Franklin, whose breakthrough X-ray diffraction data was used to
formulate the DNA structure, died in 1958, and thus was ineligible to be
nominated for a Nobel Prize.)
Nucleic acid hybridization
Hybridization is the process of complementary base 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. T and A rich regions are more easily melted than C and G rich regions. Some base steps (pairs) are also susceptible to DNA melting, such as T A and T G. These mechanical features are reflected by the use of sequences such as TATA at the start of many genes to assist RNA polymerase in melting the DNA for transcription.
Strand separation by gentle heating, as used in polymerase 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. The cell avoids this
problem by allowing its DNA-melting enzymes (helicases) to work concurrently with topoisomerases, which can chemically cleave the phosphate backbone of one of the strands so that it can swivel around the other. Helicases unwind the strands to facilitate the advance of sequence-reading enzymes such as DNA polymerase.
Base pair geometry
Base pair geometries
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 the normal 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:
- Shear
- Stretch
- Stagger
- Buckle
- Propeller: rotation of one base with respect to the other in the same base pair.
- Opening
- Shift: displacement along an axis in the base-pair plane perpendicular to the first, directed from the minor to the major groove.
- Slide: displacement along an axis in the plane of the base pair directed from one strand to the other.
- Rise: displacement along the helix axis.
- Tilt: rotation around the shift axis.
- Roll: rotation around the slide axis.
- Twist: rotation around the rise axis.
- x-displacement
- y-displacement
- inclination
- tip
- pitch: the height per complete turn of the helix.
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.
Note that "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".
Helix geometries
At least three DNA conformations are believed to be found in nature, A-DNA, B-DNA, and Z-DNA. The B form described by James Watson and Francis Crick is believed to predominate in cells. It is 23.7 Å wide and extends 34 Å per 10 bp
of sequence. The double helix makes one complete turn about its axis
every 10.4–10.5 base pairs in solution. This frequency of twist (termed
the helical pitch) depends largely on stacking forces that each base exerts on its neighbours in the chain. The absolute 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 in crystallographic experiments, and in hybrid pairings of DNA and RNA strands, but DNA dehydration does occur in vivo, and A-DNA is now known to have biological functions. Segments of DNA that cells have been methylated
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, L-DNA (the enantiomeric form of D-DNA), P-DNA, S-DNA, Z-DNA, etc. have been described so far. In fact, only the letters F, Q, U, V, and Y are now available to describe any new DNA structure that may appear in the future.
However, most of these forms have been created synthetically and have
not been observed in naturally occurring biological systems. There are also triple-stranded DNA forms and quadruplex forms such as the G-quadruplex and the i-motif.
The structures of A-, B-, and Z-DNA.
The helix axis of A-, B-, and Z-DNA.
| 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) |
Grooves
Major and minor grooves of DNA. Minor groove is a binding site for the dye Hoechst 33258.
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 a binding site.
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.
The narrowness of the minor groove means that the edges of the bases
are more accessible in the major groove. As a result, proteins like transcription 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. 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.
Non-double helical forms
Alternative non-helical models were briefly considered in the late 1970s as a potential solution to problems in DNA replication in plasmids and chromatin. However, the models were set aside in favor of the double-helical model due to subsequent experimental advances such as X-ray crystallography of DNA duplexes and later the nucleosome core particle, and the discovery of topoisomerases. Also, the non-double-helical models are not currently accepted by the mainstream scientific community.
Single-stranded nucleic acids (ssDNA) do not adopt a helical formation, and are described by models such as the random coil or worm-like chain.
Bending
DNA is a relatively rigid polymer, typically modelled as a worm-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.
Persistence length, axial stiffness
| 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:
The length of DNA over which the time-averaged orientation of the polymer becomes uncorrelated by a factor of e.
This value may be directly measured using an atomic force microscope
to directly image DNA molecules of various lengths. In an aqueous
solution, the average persistence length is 46–50 nm or 140–150 base
pairs (the diameter of DNA is 2 nm), although can vary significantly.
This makes DNA a moderately stiff molecule.
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 the minor and major grooves.
Models for DNA bending
| Step | Stacking ΔG /kcal mol−1 |
|---|---|
| T A | -0.19 |
| T G or C A | -0.55 |
| C G | -0.91 |
| A G or C T | -1.06 |
| A A or T T | -1.11 |
| A T | -1.34 |
| G A or T C | -1.43 |
| C C or G G | -1.44 |
| A C or G T | -1.81 |
| G C | -2.17 |
The entropic flexibility of DNA is remarkably consistent with standard polymer physics models, such as the Kratky-Porod worm-like chain model. Consistent with the worm-like chain model is the observation that bending DNA is also described by Hooke's law at very small (sub-piconewton)
forces. However, 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.
Bending preference
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 and T
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 in nucleosome particles. See base step distortions above.
DNA molecules with exceptional bending preference can become intrinsically bent. This was first observed in trypanosomatid kinetoplast DNA. Typical sequences which cause this contain stretches of 4-6 T and A residues separated by G and C 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.
Circularization
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),
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
significant energy 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.
Stretching
Elastic stretching regime
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 the thermal bath
of the solvent. This is due to the thermal vibration of the molecule
combined with continual collisions with water molecules. For entropic
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. Using optical tweezers, the entropic stretching behavior of DNA has been studied and analyzed from a polymer physics perspective, and it has been found that DNA behaves largely like the Kratky-Porod worm-like chain model under physiologically accessible energy scales.
Phase transitions under stretching
Under sufficient tension and positive torque, DNA is thought to undergo a phase transition
with the bases splaying outwards and the phosphates moving to the
middle. This proposed structure for overstretched DNA has been called P-form DNA, in honor of Linus Pauling who originally presented it as a possible structure of DNA.
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 as S-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.
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,
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 the GNC_hypothesis to be of evolutionary importance.
Supercoiling and topology
Supercoiled structure of circular DNA molecules with low writhe. The helical aspect of the DNA duplex is omitted for clarity.
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
negatively supercoiled. DNA in vivo is typically negatively supercoiled, which facilitates the unwinding (melting) of the double-helix required for RNA transcription.
Within the cell most DNA is topologically restricted. DNA is typically found in closed loops (such as plasmids
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.
Analysis of DNA topology uses three values:
- L = linking number - the number of times one DNA strand wraps around the other. It is an integer for a closed loop and constant for a closed topological domain.
- T = twist - total number of turns in the double stranded DNA helix. This will normally tend to approach the number of turns that a topologically open double stranded DNA helix makes free in solution: number of bases/10.5, assuming there are no intercalating agents (e.g., ethidium bromide) or other elements modifying the stiffness of the DNA.
- W = writhe - number of turns of the double stranded DNA helix around the superhelical axis
- L = T + W and ΔL = ΔT + ΔW
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 are topologically 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 termed topoisomerases.
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 of recombination in linear DNA which have similar topological constraints.
The linking number paradox
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". However, when experimentally determined structures of the nucleosome displayed an over-twisted left-handed wrap of DNA around the histone octamer, this paradox was considered to be solved by the scientific community.
