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The structural basis of DNA flexibility

A. A. Travers
Phil. Trans. R. Soc. Lond. A 2004 362, 1423-1438
doi: 10.1098/rsta.2004.1390

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10.1098/rsta.2004.1390

The structural basis of DNA exibility

By A. A. Travers
MRC Laboratory of Molecular Biology, Hills Road,
Cambridge CB2 2QH, UK (aat@mrc-lmb.cam.ac.uk)
Published online 13 May 2004

Although the average physico-chemical properties of a long DNA molecule may

approximate to those of a thin isotropic homogeneous rod, DNA behaves more
locally as an anisotropic heterogeneous rod. This bending anisotropy is sequence
dependent and to a rst approximation reects both the geometry and stability of
individual base steps. The biological manipulation and packaging of the molecule
often depend crucially on local variations in both bending and torsional exibility. However, whereas the probability of DNA untwisting can be strongly correlated with a high bending exibility, DNA bending, especially when the molecule is
tightly wrapped on a protein surface, may be energetically favoured by a less exible
sequence whose preferred conguration conforms more closely to that of the complementary protein surface. In the latter situation the lower bending exibility may
be more than compensated for on binding by a reduced required deformation energy
relative to a fully isotropic DNA molecule.
Keywords: bending exibility; torsional exibility; anisotropic bending;
nucleosome; TpA base step; genomic distribution of exible sequences

1. Introduction
The early representations of DNA presented the molecule as a static straight rod
(de Chadarevian & Kamminga 2002). Yet in reality the DNA double helix is a highly
dynamic structure that can both bend and twist. This exibility can be described
by two parameters: torsional exibility, characterized by alterations in the twist
angle between adjacent base pairs, and bending exibility, in which the long axis
of the double helix deviates, both locally and globally, from a straight trajectory.
These properties depend primarily on the physico-chemical properties of individual
base steps and to a lesser extent on the DNA sequence context in which they are
embedded. Since the physical characteristics of the 10 possible base pairs vary substantially, the exibility of DNA is a sequence-dependent property and varies both
locally, depending on the presence of a particular sequence, and globally, depending
on the overall base composition of the molecule in question. The major determinants
of exibility are the stacking energy of a given base step, the number of hydrogen
bonds in a base pair, and the occupancy of both the major and minor grooves by
exocyclic groups. Only the last parameter imparts directionality or anisotropy to
bending exibility and together with the dipole on the contiguous base pairs denes
the conformational space available to a base step.
One contribution of 16 to a Theme The mechanics of DNA.
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c 2004 The Royal Society

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A. A. Travers

Both the torsional and bending exibility of DNA have important biological consequences. The processes of transcription and DNA replication both require that
the DNA be untwisted prior to the initiation of the copying of the DNA sequence.
This untwisting is often initiated at particularly thermodynamically labile sequences
(Drew et al . 1985). Bending exibility is also an essential aspect of biological function. In all living cells the DNA genome is packaged in a small volume and the
necessary compaction is achieved by the tight bending of DNA around a protein
scaold. The archetypal example of this packaging in the eukaryotic nucleus is
the wrapping of 145 bp of DNA in 1.67 toroidal superhelical turns of radius 43
A
around the histone octamer to form a nucleosome core particle (equivalent maximally to ca. 5060 per double helical turn) (Luger et al . 1997; Richmond & Davey
2003). Similarly, the manipulation of DNA during transcription or DNA recombination often requires the tight bending of DNA around the appropriate protein
assembly.

2. Bending exibility
At the zero level of approximation the exibility of a DNA molecule can be modelled in terms of a thin isotropic homogeneous rod in which the DNA can bend in
any direction (Bloomeld et al . 1974). However, the biological consequences of the
physico-chemical properties of a DNA molecule often depend crucially on relatively
small sequence-dependent dierences in the energetics of bending or untwisting. Consequently, for the packaging or enzymatic manipulation of DNA in vivo, the secondorder approximation model of DNA as an anisotropic heterogeneous rod (Calladine
& Drew 1986; Travers & Klug 1987; Sivolob & Khrapunov 1995) in which the DNA
has a preferred direction of bending is more appropriate for descriptions of bending exibility. These properties in turn are the summation of the properties over all
individual base steps in any sequence.
The bending exibility of DNA is conventionally described in terms of its inverse,
stiness, which is expressed as the persistence length, P , a common measure of polymer bending rigidity. P is dened as the length over which the average deection
of the polymer axis caused by thermal agitation is 1 rad. A number of techniques
including light scattering (Sobel & Harpst 1991), rotational diusion (Hagerman
1988), DNA cyclization kinetics (Shore & Baldwin 1983; Taylor & Hagerman 1990),
cryo-electron microscopy (Bednar et al . 1995), as well as conventional electron microscopy (Frontali et al . 1979) and atomic-force microscopy (Berge et al . 2002)have
led to estimates for P of ca. 50 nm for mixed-sequence B-form DNA. Below this
length the DNA behaves on average in solution essentially as a sti rod. However,
the measured persistence length, as determined by most techniques, especially those
that use visualized trajectories of DNA molecules, depends not only on the intrinsic
exibility of the DNA molecule but also on its intrinsic curvature, i.e. on the isotropy
of the exibility (Bednar et al . 1995). Only when exibility is fully isotropic, i.e. the
molecules are intrinsically straight, do measurements of P give an accurate measure
of the intrinsic exibility.
DNA is both polymorphic and heterogeneous in sequence. Both these parameters
strongly inuence stiness and are relevant to the biological role of DNA exibility.
Another signicant factor is the ionic environment, which can modulate the repulsion
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The structural basis of DNA exibility

(a)
y-displacement
z
y
x

1425

(b)
x-displacement

shift

slide

inclination

roll

tilt

rise

translations
tip

twist

rotations
Figure 1. Schematics of the (a) base pair and (b) base pair step parameters of the DNA double
helix. The arrows for the base pair parameters indicate positive translational and rotational
displacements. The major groove is a positive x. (Adapted from Dickerson et al . (1989).)

between the phosphate backbones across the major and minor grooves. In triuoroethanol the persistence length of A-form DNA is about threefold greater than that
of B-DNA in a normal aqueous environment (Charney et al . 1991). However, in the
presence of ethanol, which also promotes the B-form to A-form transition, DNA
becomes more exible (Fang et al . 1999).
Base composition and sequence are also critical determinants of DNA exibility.
Comparison of the Young modulus of synthetic polymers of dierent base composition showed that although in general stiness is correlated with G/C content the
nature of the base steps present in polymers of the same composition can signicantly inuence stiness (Hogan et al . 1983; Hogan & Austin 1987). In particular,
the homopolymers (dR:dT) have a longer persistence length than the corresponding copolymers d(RY):d(RY). In these experiments the sequence repeats are short
relative to the helix repeat and so the molecules can be regarded as intrinsically
straight.

3. Chemical determinants of DNA exibility

The ability of a base step to be distorted depends on several properties. Among
the most important of these are the electronic congurations of its component base
pairs, the number of intra- and inter-base pair H-bonds and the presence of exocyclic
groups in the major and minor grooves. Together these characteristics determine two
important parameters: the range of conformations or conformational space that can
be readily adopted by a particular step, and the deformation energy necessary to
adopt a particular conformation. The conformational space may be regarded as a
local determinant of isotropy in the sense that a step that can adopt a wide range of
conformations can change the direction of the double helical axis over a correspondingly wide range, whereas any change conferred by a base step with only a limited
conformational space will be restricted to a narrow range.
The bending of the DNA double helix implies that locally the trajectory of the
helical axis is changed. Such changes depend on the particular geometry of the relevant base steps. On the outside of a bend the DNA grooves, both major and minor,
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1426

A. A. Travers
(a)

H
N

C'

Me

N
C'

O
(b)
H
H

C'

N
H

C'

H
Figure 2. Electronic congurations of (a) an AT and (b) a GC base pair showing the
resultant dipole moment. (Reproduced with permission from Hunter (1996).)

sd ()

CA
(=TG)

GG(=CC),
GC, CG
GA
(=TC)
AA
(=TT)

AT
AC
(=GT)

TA
GG(=CC), GC, CG
3

20

10

10

20

roll (deg)

55

45

35

25

25

helical twist (deg)

Figure 3. Ranges of conformational space occupied by dierent dinucleotide steps in crystal

structures of DNA oligomers. Note the coupling of twist, roll and slide (sd). (Adapted and
reproduced with permission from El Hassan & Calladine (1997).)

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The structural basis of DNA exibility

1427

must be wider to accommodate the curvature, while on the inside they will be narrower. In principle, changes in the direction of the helical axis can result from two
main types of displacement of one base pair relative to the other in a single base
step. These are roll, in which one base pair is rotated about the long axis relative
to the other, and tilt, in which the rotational displacement is about the short axis
(gure 1). Another important variable in the geometry of base steps is slide, which
is the displacement of one base pair relative to its neighbour along the direction of
the long axis of a dinucleotide step. Shift is the corresponding displacement along
the short axis. In crystal structures of DNA oligomers the parameters roll, slide and
twist are often correlated as also are tilt and shift (El Hassan & Calladine 1997).
Thus negative roll correlates with high twist and positive slide and vice versa (gure 3).
Both for free and protein-bound DNA the predominant deformation associated
with bending is roll. This is energetically more favourable than the tilt rotation and
alters groove width in the direction of curvature. Thus a base step with positive roll
opens the minor groove (and correspondingly closes the major groove) at a position
where the minor groove is on the outside of the bend. Similarly, a base step with
negative roll narrows the minor groove. When DNA is wrapped tightly on a protein
surface, as in the nucleosome core particle, it is at precisely these positions where
the base-step deformation is greatest and hence conformationally rigid steps may be
disfavoured.
In free DNA the preferred conformations of base steps are determined by van der
Waals and electrostatic interactions between adjacent base pairs (Calladine 1982;
Yanagi et al . 1991; Hunter 1993; Young et al . 1995; Gorin et al . 1995; El Hassan
& Calladine 1997). The electrostatic interactions are dominated by a large dipole
on GC pairs contrasted with a diuse distribution of charge on AT base pairs
(Hunter 1993, 1996; Gallego et al . 1995) (gure 2). A major consequence of these
dierent distributions is that whereas adjacent AT base pairs can stack without
displacement due to electrostatic forces, the optimum conguration for adjacent GC
base pairs is one in which the otherwise repulsive dipoles are accommodated by a
slide displacement with a consequent tendency to a higher positive roll angle.
Another factor that inuences the compressibility of both the major and minor
grooves and hence the general bending exibility is the presence of exocyclic groups
in the grooves. To test both the general notion that DNA exibility is a determinant on nucleosome core particle formation and the particular hypothesis that the
exocyclic purine 2-amino group in the minor groove directly aects the persistence
length, selected high-anity histone octamer binding sequences were substituted
with either hypoxanthine for guanine or diaminopurine for adenine, substitutions
which respectively remove or add a purine 2-amino group. Using visualization by
atomic-force microscopy to determine an apparent persistence length P  , where P  is
the measure incorporating both intrinsic curvature and true exibility, P  was shown
to be directly correlated with the exocyclic load in the minor groove (Virstedt et al .
2004). Notably in the diaminopurine-substituted DNA, in which all the base pairs
possess a purine 2-amino group, the rise per base pair was reduced from the 3.35
A
characteristic of B-DNA to ca. 3.0
A, a value that implies a shift towards A-form
DNA. Such a shift would be consistent with a decreased compressibility of the minor
groove.
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A. A. Travers

4. The sequence dependence of DNA bending

The conformational analysis of the structures of individual base steps is largely
derived from crystal structures of DNA oligomers. In these crystals the DNA adopts
both B-type and A-type congurations and possibly because of crystal packing forces
the conformation of a sequence in the crystal may dier from that in solution as determined by NMR (Dornberger et al . 1998). The range of conformations adopted by
specic base steps in free DNA has been analysed from a compilation of the characteristics of base steps in such crystal structures (El Hassan & Calladine 1997).
The conclusion from these studies, which considered all crystal structures containing the canonical bases, is that certain steps, notably AA/TT, AT and GA/TC,
occupy a rather restricted region of conformational space (gure 3). All these base
steps exhibit a preference for low slide/low roll. By contrast, other steps, particularly AC/GT, TA and TG/CA, occupy a larger continuous domain of conformational
space. Of these three steps TG/CA shows the greatest variation in conformation. The
three G/C-containing base steps, GC, CG and GG/CC, are all bistable, exhibiting
preferred conformations with either high or low, but not intermediate, slide. A correlation between high propeller twist and conformational rigidity was also noted
(El Hassan & Calladine 1996). When this type of analysis is restricted to crystals
where the DNA has adopted the B-form, as dened by sugar conformation, the base
steps TA and TG/CA are the most variable, and hence most exible, while AT,
GA/TC and AA/TT are the least variable (Neugebauerova & Kypr 2000). By contrast, in A-DNA crystals, TA, TG/CA and AC/GT, together with AT, exhibit the
least, and AA/TT together with AG/CT the greatest conformational variability.
By these criteria of allowed conformational space, AA/TT and AT are among the
least, and TG/CA together with TA, the most exible dinucleotide steps in B-type
DNA in solution. For the TA and TG/CA steps this accords with the experimental
nding that these steps have the lowest stacking energies (E. Protozanova & M. D.
Frank-Kamenetskii 2004, personal communication). However, relative to other steps,
AA/TT and AT have only intermediate stacking energy while GC has the highest. An
additional measure of conformational constraint, or rigidity, is the ability of certain
short sequences to confer intrinsic curvature when present in helical register. This
ability is dependent not only on the preferred conformation of the particular base
step but also on its sequence context. Thus isolated AA or TT dinucleotides do not
confer signicant intrinsic curvature but oligo (dA:dT)n runs, where n = 3, do (Koo
et al . 1986; Diekmann 1992). Similarly, short G/C-rich sequences, notably GGCC,
can confer intrinsic curvature but only when the narrow major groove is stabilized
by divalent cations (Brukner et al . 1993).

5. Bending and torsional exibility

There is, however, a crucial dierence between the sequence dependence of DNA
wrapping and untwisting. The energetics of untwisting are strongly favoured by the
presence of TA base steps (Drew et al . 1985). Although these steps also favour
DNA wrapping (Rhodes 1979; Lowary & Widom 1998; Fitzgerald & Anderson 1999)
an anisotropically exible DNA sequence, which is necessarily more rigid than an
isotropically exible sequence of the same base composition, can promote wrapping
both because the entropic penalty on binding may be substantially lower (Anselmi
et al . 1999) and because the deformation required for wrapping may be less.
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The structural basis of DNA exibility

1429

In general, there is a positive correlation between the persistence length of a DNA

molecule and its bulk melting temperature (Tm ). Since Tm is a direct measure of
torsional rigidity it follows that for a particular sequence the bending and torsional
exibility of DNA are related and that both are manifestations of the summation
of the conformational exibilities of individual base steps in their sequence context.
However, this correlation is not maintained when dierent sequences are compared.
In general, Tm is directly correlated with the purine 2-amino group content and
the corresponding frequency of base pairs with three hydrogen bonds, regardless
of sequence, but the persistence length depends much more strongly on particular
sequence contexts. Although the substitutions aect the number of hydrogen bonds in
a base pair they do not qualitatively alter the type of base steps (purinepyrimidine,
purinepurine or pyrimidinepurine) and their contained steric interactions. These
interactions are major determinants of bending exibility (El Hassan & Calladine
1996).
In an earlier study, Sivolob & Khrapunov (1995) compared the persistence lengths
of synthetic polymers with melting temperatures reecting the energy of base stacking within a given step (Gotoh & Tagashira 1981; Vologodskii et al . 1984). They
observed that although there was a general correlation between persistence length
and Tm , there were also signicant deviations from a good t to linearity. In particular, the synthetic polymers poly(dG:dC) and poly(dGC:dGC) were respectively more
and less rigid than would have been predicted from a strict correlation between persistence length and Tm . These molecules have the same base composition and hence
the same purine 2-amino group content but dier in the type of base steps. Thus
although the Tm of a base step is an important determinant of bending exibility,
the ability of particular base steps to adopt alternative conformations (El Hassan &
Calladine 1997) may also contribute substantially. In general, any correlation, albeit
imperfect, between bending exibility and Tm , is probably consequent on stacking
energy, rather than base pairing, being the major determinant of base step stability (SantaLucia 1998; E. Protozanova & M. D. Frank-Kamenetskii 2004, personal
communication).

6. Protein-induced DNA bending

The local deformation of DNA by DNA-binding proteins does in many cases substantially exceed the normal conformational uctuations of free DNA due to thermal
motion in solution. For example, proteins containing an HMG domain can induce a
bend of 90110 in a binding site of 78 bp, while in the nucleosome core particle the
apparent average curvature is ca. 47 per double helical turn. Protein-induced DNA
wrapping is thus a test case for the general postulate that, in the absence of direct
sequence-specic recognition, the bending exibility of DNA determines its anity
for the complementary protein surface. Alteration of the persistence length of specic DNA sequences by substituting hypoxanthine for guanine or diaminopurine for
adenine shows that there is, in general, a strong inverse correlation between persistence length and nucleosome formation (Virstedt et al . 2004). The only exception
to this correlation occurs when the base substitution, although decreasing P  , at
the same time increases the isotropy of the DNA exibility and so imposes a larger
entropic penalty for binding by the histone octamer. In addition to the eects of
altering the content of the purine 2-amino group in the minor groove, comparable
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1430

A. A. Travers
50
dyad
45
40
AA/TT
35
N
30
25

10
GC

10

20

30
40
50
assigned position in core

60

70

Figure 4. Variations in the occurrence of the dinucleotides AA/TT and GC versus position in the
nucleosome core DNA sequence. N is the running 3-bond average of the frequency of occurrence
of a dinucleotide also averaged about the dyad at the central base step 72.5. (Data taken from
Satchwell et al . (1986).)

alterations in the major groove which change the content of the pyrimidine 5-methyl
group, i.e. substitution of uracil for thymine and of 5-methyl cytosine for cytosine,
also respectively increase or reduce the anity for the histone octamer (Buttinelli et
al . 1998).
Although the extent of DNA bending in such proteinDNA complexes is substantially greater than that in free DNA, the preferred positioning of base steps in the
tightly wrapped DNA accords remarkably well with the geometrical properties of the
same base steps as deduced from crystal structures. In the nucleosome core a major
determinant of the rotational placement of the DNA is the periodic occurrence of
short A/T-rich sequences in one helical phase, and of short G/C-rich sequences in the
opposite phase (gure 4). These various short sequences favour local conformations
with narrow or wide minor grooves, respectively, and thus together facilitate the tight
wrapping of DNA (Satchwell et al . 1986; Calladine & Drew 1986; Shrader & Crothers
1990). A similar pattern is observed in the much shorter binding site of the CAP transcription factor, which bends DNA by ca. 90 (Travers & Klug 1987; Gartenberg &
Crothers 1988). The sequences with these preferential placements are generally precisely those whose conformational exibility in crystals of B-type DNA is restricted
to a narrow range. The periodic pattern therefore arises because these conformationally rigid sequences are preferentially excluded from positions where they are
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The structural basis of DNA exibility

1431

forced to adopt a highly energetically unfavourable conformation. However, two base

steps which are conformationally exible can nevertheless act as strong positioning
signals. These are the pyrimidinepurine steps CA/TG, which uniquely of the 10 possible dinucleotides is located preferentially at both inward- and outward-facing minor
grooves but not in between, and TA, which is located at inward-facing minor grooves
in the strongest positioning sequences (Lowary & Widom 1998; Th
astrom et al . 1999;
Roychoudhury et al . 2000; Widlund et al . 1999). By contrast, other short sequences
which can readily adopt a wide range of conformations have, when repeated, a high
anity for the histone octamer but do not direct precise positioning. Thus, some
short sequences in isolation show almost no preference for a preferred rotational
position in nucleosomal DNA. The most notable example of such a sequence is the
trinucleotide CAG/CTG, which has no rotational preference in mixed-sequence core
particles (Satchwell et al . 1986) but which, when present in multiple repeats, has
an unusually high anity for the histone octamer (Wang & Grith 1995). This
same trinucleotide is also the one which is the most frequently cleaved by DNase I
in mixed-sequence DNA and by this criterion and others is deemed to be the most
exible (Brukner et al . 1995; Chastain & Sinden 1998).
Signicant insights into the structural deformations necessary for the wrapping
of DNA on the surface of the histone octamer have recently become apparent in a
crystal structure solved to 1.9
A resolution of a nucleosome core particle containing
147 bp DNA derived from a satellite DNA sequence that positions nucleosomes precisely (Richmond & Davey 2003). The average conformation of the wrapped DNA
approximates closely to that of B-form DNA from bre studies with an average rise
between base pairs of 3.36
A and an average propeller twist of 9 . Relative to DNA
in solution with 10.510.6 bp per turn, the DNA in the core particle is slightly overtwisted, the overall average twist value in the laboratory reference frame (Travers
& Klug 1987), i.e. the intrinsic twist, as calculated from successive 10 bp segments,
being 34.52 , corresponding to 10.43 bp per turn. This latter value is equivalent to
an average structural periodicity of 10.30 bp per turn in the local reference frame, in
excellent agreement with the structural periodicities obtained from earlier sequence
analysis (Drew & Travers 1985; Satchwell et al . 1986), and also from the modulation of pyrimidine dimer formation and hydroxyl-radical cleavage (Gale et al . 1987;
Hayes et al . 1990). The DNA wrapping is thus accomplished primarily within the
normal constraints of B-DNA conformation and is achieved by local conformational
variation.
The DNA groove dimensions exhibit a regular periodicity throughout the nucleosome core. The minor-groove width varies from 3
A at the proteinDNA contact sites
to 9
A where the major groove faces the protein. There are corresponding variations
in the depth of the groove between 7 and 3
A. This periodic variation in conformation
is also reected in the twist, since signicant overtwisting seems to be associated with
a narrow minor groove occurring at all the anchor points. However, in the crystal
structure there is no obvious dependence of groove width on the underlying DNA
sequence, except that in an overall sense, the entire DNA sequence has somehow
made a best t with the curved surface of the protein; in other words, the positioning is dependent on the ensemble of deformation energies necessary to wrap the
DNA.
Because the deection of the helical axis is greater, the deformations of the base
steps are greater and in some cases essentially dierent in wrapped DNA from those
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1432

A. A. Travers
(a)

(b)
smooth bending
into major groove

smooth bending
into minor groove

9.4
8
6.0 9
7.6 10
6.6 11
5.5 12
2.7

8.3
6.5
7.6
10.6
4.5
9.4

3
4
5
6
7

(c)
kinked bending
into minor groove
8.3
3.7
24.9
0.7
2.8
5.0

34
35
36
37
38

Figure 5. DNA bending in nucleosome core DNA. Structures (left) and schematic representations
(right) stress uniformity of curvature in the major-groove blocks (red) (a) and alternating shift
values (b) and localization of curvature in kinks in minor-groove blocks (c) (yellow for one
representative double-helical turn). Also indicated are the primary bound-phosphate groups
(green), the block-junction phosphate groups (white) and the DNA axes for the actual (gold) and
ideal (white) superhelices. The contributions of base pair step curvature to superhelix bending
are listed with base pair numbers (centre). (Adapted from Richmond & Davey (2003) with
permission.)

in free DNA. In the nucleosome core the roll component of the base steps contributes
three times as much as the tilt component to the curvature of the superhelix (Richmond & Davey 2003). Curvature into the superhelix is therefore accumulated every
ve base pairs, where either the minor or the major groove faces the histone octamer,
again in excellent agreement with the sequence preferences in these positions. However, there is poor correlation between the deformations observed in the particular
sequence studied and the sequence preferences characteristic of a population of nucleosome core particles containing dierent DNA sequences.
The nature of deformations required for bending diers in dierent regions of
the nucleosome core. In the central region where the DNA contacts a tetramer of
histones H3 and H4 bending is smooth: that is, the deformations are spread over
several adjacent base steps. By contrast, where the DNA contacts each of the histone
H2A/H2B dimers bending can be discontinuous with the bending arising from a large
deformation, or kink, at a single base step. Smooth bending into the major groove
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The structural basis of DNA exibility

1433

excess roll
+24
PPa

24

outside

3'

5'
tip

inside
12

+12

PPa
Figure 6. Oscillation of the base pairtip parameter for nucleosome core DNA. Coupling between
PPa , tip and excess roll. PPa is the component of the phosphatephosphate distance that lies in
the plane containing the local superhelical path. Rotation of a base pair along its tip axis (red)
in the direction indicated decreases PPa on the inside and increases it on the outside of the
superhelix. Excess roll for a base pair step is the dierence between the tip angles for the two
contributing base pairs. The DNA blocks shown are from the central region of the superhelix.
(Adapted from Richmond & Davey (2003) with permission.)

is characterized by a uniform positive roll angle and undertwisting at adjacent base

steps. By contrast, smooth bending into the minor groove combines a relatively
constant negative roll angle with an alternating twistshift coupling which relieves
the steric hindrance between the base edges (gure 5). In the sequence studied, the
discontinuous bending into the minor groove is accomplished entirely by kinking of
CA/TG base steps with roll angles of 18 to 27 and a coupled overtwisting. This
ability to kink and overtwist explains the unusual positioning properties of both the
CA/TG and TA base steps. The stacking energy of both steps is low and both can
adopt either high or low roll angles by reducing the stacking energy. However, the
unstacking energy is substantially less for TA than for CA/TG (E. Protozanova &
M. D. Frank-Kamenetskii 2004, personal communication) and consequently TA is
the stronger positioning signal, especially at positions where the bending is greatest,
for example ca. 15 bp on either side of the dyad (Fitzgerald & Anderson 1999). It
is notable that for a strong positioning sequence which contains a nearly vefold
excess of CA/TG base steps over TA steps (49 and 10, respectively) the TA steps
are located preferentially at inward-facing minor grooves (Virstedt et al . 2004). The
positioning of a histone octamer on a DNA sequence is thus dependent both on the
exclusion of short inexible sequences such as GGC or AAA/TTT from unfavourable
rotational positions and from the selective placement of highly deformable steps at
positions where the DNA curvature is greatest. Both these processes act to minimize
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A. A. Travers

the overall deformation energy and determine rotational and, especially in the latter
case, translational positioning.
An unexpected feature of the structure of wrapped nucleosomal DNA is the oscillation of the conformational parameter tip (gure 6) along the length of the DNA. The
oscillation has two structural consequences. It increases the total scalar curvature due
to wrapping and crucially changes the component of the phosphatephosphate distance lying parallel to the path of the superhelix. This distortion thus accommodates
the substantial dierence in arc length/double-helical turn between the inside and
the outside of the superhelix (gure 6).

7. DNA exibility and genomic organization

To what extent are variations in DNA exibility reected in genomic organization?
An excellent example of the dependence of biological function on DNA structure is
provided by the genome of the enteric bacterium Escherichia coli. In this organism
the strongest promoters for DNA transcription are almost invariably associated with
A/T-rich, and hence exible, DNA sequences extending upstream for 100300 base
pairs from the transcription start point (Pedersen et al . 2000). The activity of many
of these promoters is strongly dependent on a high negative superhelical density
stored in the DNA. This would in principle favour both DNA untwisting at sequences
such as TATAAT close to the start point (Drew et al . 1985) and also left-handed
DNA wrapping around the protein complex responsible for initiating transcription.
In many of these highly active promoters the DNA sequence also imparts curvature
to the region, a feature that correlates both with the presence of multiple binding
sites for the abundant DNA bending protein FIS (factor for inversion stimulation)
and the constraint of negative superhelicity in the protein complex (Rochman et al .
2002). In other such promoters an alternative model proposes that the upstream
activating sequence contains regions that are highly susceptible to DNA untwisting
(Hateld & Benham 2002). For both DNA wrapping and untwisting in the upstream
region both models predict that the topological unwinding is transmitted to the
TATA sequence and promotes its untwisting.
Most of the information available on the organization of nucleosome arrays has
been obtained either from in vitro studies or from in vivo characterization of localized
regions. However, the availability of genome sequences has enabled the analysis of
the periodicity of occurrence of di- or trinucleotides within large stretches of genomic
DNA. Analysis of human exon sequence by a 10-state Markov model revealed a
preference for in-phase occurrence of the trinucleotide CAG and its complement CTG
with weaker preferences for AAG, GAG, ATG and GTG (Baldi et al . 1996). All these
trinucleotides, with the exception of AAG, are strongly cut by DNase I (Brukner et al .
1995) and thus exible in the sense that they can adopt a conformation characteristic
of an outward-facing minor groove on nucleosomal DNA (see Hogan et al . 1989).
However, this same analysis did not detect any preferred occurrence of trinucleotides
in the opposite phase corresponding to sequences, such as AAA/TTT, which are
preferentially located at inward-facing minor grooves in the nucleosome core particle.
In marked contrast to this result an analysis of the genome sequences of Saccharomyces cerevisiae and Caenorhabditis elegans by the generation of Fourier power
spectra showed that several dinucleotides, of which AA/TT yielded the strongest
signal, produced peaks at a periodicity of ca. 10.2 bp (Widom 1996). These peaks
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The structural basis of DNA exibility

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were unique to eukaryotic rather than prokaryotic genomes. Other dinucleotides of

signicant signal strength which were observed at this periodicity were GG/CC,
GC and GA/TC. Of these four dinucleotides, AA/TT, GG/CC and GC, but not
GA/TC, have all been shown previously to occupy preferred rotational positions in
core nucleosomal DNA (Satchwell et al . 1986). This correlation between the two sets
of data strongly indicates that the in vivo periodicities may reect sequence constraints imposed by nucleosomal organization and therefore that the signals characterized for rotational positioning in vitro are also observed in vivo and constitute a
signicant element of the genomic organization of DNA sequences.

8. Concluding remarks
Variations in the local conformational exibility of DNA facilitate not only the packaging of the molecule on nucleosomes and its consequent compaction but also targeted untwisting during transcription initiation. This variation is reected in the
genomic organization of DNA sequences and complements the role of DNA as an
informational macromolecule.

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