Báo cáo khoa học: C fi G base mutations in the CArG box of c-fos serum response element alter its bending flexibility Consequences for core-SRF recognition potx

Ana-lysis by singular value decomposition of a set of Raman spectra recorded as a function of temperature, revealed a premelting transition associated with a conformational shift in the

response element alter its bending flexibility

Consequences for core-SRF recognition

Josef Stepanek1,2,*, Michel Vincent3, , Pierre-Yves Turpin1, Denise Paulin2, Serge Fermandjian4,5, Bernard Alpert2and Christian Zentz1

1 Laboratoire de Biophysique Mole´culaire Cellulaire & Tissulaire, Universite´ Pierre et Marie Curie, Evry, France

2 Laboratoire de Biologie Mole´culaire de la Diffe´renciation, Universite´ Denis Diderot, Paris, France

3 LURE, Universite´ Paris-Sud, Orsay, France

4 De´partement de Biologie et Pharmacologie Structurales, Ecole Normale Supe´rieure de Cachan, France

5 Institut Gustave Roussy, Villejuif, France

Specific binding of the serum response factor (SRF) to

the serum response element (SRE) requires a consensus

sequence CC(A⁄ T)6GG, the CArG box [1–7] The

transcriptional activity of a number of

CArG-depen-dent genes is associated with SRF-binding activity

[8–14] The c-fos gene contains a single high-affinity CArG box, whereas many muscle-specific genes contain two or more CArG boxes However, these carry substitutions with G or C nucleotides within their (A⁄ T) domain, thus lowering the affinity [15–19]

Keywords

CArG box; c-fos; DNA bending; DNA

dynamics; serum response element

Correspondence

C Zentz, Laboratoire de Biophysique

Mole´culaire Cellulaire & Tissulaire,

Universite´ Pierre et Marie Curie, CNRS

UMR 7033, GENOPOLE Campus 1, 5 rue

Henri Desbrue`res, 91030 Evry Cedex,

France

Fax: +33 1 69 87 43 60

Tel: +33 1 69 87 43 52

E-mail: zentz@ccr.jussieu.fr

*Permanent address

Charles University, Faculty of Mathematics

and Physics, Prague, Czech Republic

 Present address

IBBMC, Universite´ Paris-Sud, Orsay France

(Received 22 December 2006, revised 20

February 2007, accepted 2 March 2007)

doi:10.1111/j.1742-4658.2007.05768.x

By binding to the CArG box sequence, the serum response factor (SRF) activates several muscle-specific genes, as well as genes that respond to mitogens The core domain of the SRF (core-SRF) binds as a dimer to the CArG box C)5C)4A)3T)2A)1T+1T+2A+3G+4G+5 of the c-fos serum response element (SREfos) However, previous studies using 20-mer DNAs have shown that the binding stoichiometry of core-SRF is significantly altered by mutations C)5fi G (SREGfos) and C)5C)4fi GG (SREGGfos)

of the CArG box [A Huet, A Parlakian, M-C Arnaud, J-M Glandie`res, P Valat, S Fermandjian, D Paulin, B Alpert & C Zentz (2005) FEBS J 272, 3105–3119] To understand these effects, we carried out a comparative ana-lysis of the three 20-mer DNAs SREfos, SREGfos and SREGGfosin aqueous solution Their CD spectra were of the B-DNA type with small differences generated by variations in the mutual arrangement of the base pairs Ana-lysis by singular value decomposition of a set of Raman spectra recorded

as a function of temperature, revealed a premelting transition associated with a conformational shift in the DNA double helices from a bent to a linear form Time-resolved fluorescence anisotropy shows that the fluores-cein reporter linked to the oligonucleotide 5¢-ends experiences twisting motions of the double helices related to the interconversion between bent and linear conformers The three SREs present various bent populations submitted, however, to particular internal dynamics, decisive for the mutual adjustment of binding partners and therefore specific complex formation

Abbreviations

core-SRF, core domain of the serum response factor; SRE, serum response element; SRF, serum response factor; SVD, singular value decomposition.

Strong-affinity CArG boxes are likely to bind SRF

constitutively and genes appear to be regulated

primar-ily during the post-SRF binding step, owing to

interac-tions with accessory proteins [20] Weaker affinity

CArG boxes may offer additional control through a

mechanism that influences SRF binding, i.e by mutual

combined interactions of CArG boxes and accessory

proteins [21,22]

The core domain of the SRF (core-SRF) binds to

the CArG box as a homodimer [7,23,24] The specific

core-SRF–SREfoscomplex is characterized by the

par-ticular properties of the minor groove in the (A⁄ T)

domain and its flanking G:C base pairs The SRF

causes the SRE to bend  70 The role of this

bend-ing in specific recognition has been emphasized

[23,25,26] The efficiency and specificity of

SRF-depend-ent transcription may vary due to changes in the

CArG box sequence [22] To understand the origin

of these effects this study focuses on the three 20-mer

oligonucleotides: SREfos, SREGfos and SREGGfos The

SREfos sequence, 5¢-d(GGATGTC)5C)4A)3T)2A)1

T+1T+2A+3G+4G+5ACAT)-3¢, embodies the native

CArG box of the c-fos enhancer (CArG box numbered)

[2,27], whereas SREGfos carries the single C)5fi G

mutation and SREGGfos the double C)5C)4fi GG

mutation within their CArG box A previous report has

shown that the parent SREfos bound a core-SRF

homodimer, whereas the single mutant SREGfosand the

double mutant SREGGfos bound one and four

mono-mers (on average), respectively [7] This highlights the

role of the base sequence at the border of the A⁄ T track

in the specific complex assembly and functional

organ-ization How mutations affect binding of the core-SRF

and generate a lack of defined stoichiometry is an open

question Thus, we carried out a comparative analysis of

the three oligonucleotides using Raman, CD and

fluor-escence spectroscopies in order to detect their mutual

structural, electrostatic and dynamical differences CD

and Raman are sensitive to small structural changes

[28,29] In addition, Raman scattering is a powerful

means of clearing up the various sensitivities of the

nucleic acid chains to temperature [30,31] Fluorescence

studies require a fluorophore reporter, such as

fluo-rescein, chemically fixed to the oligonucleotides The

fluorescein fluorescence signal arises from the

overlap-ping emissions of the mono- and dianionic protolytic

states [32], which are sensitive to the electric charge

distribution on the DNA Chain DNA dynamics have

been extensively studied [33–37] DNA is intrinsically

flexible, but this flexibility varies from one DNA

to another [38] To date, little is known about the

relationships between the ability of DNA to bend and

its effects on protein binding Previous studies have

shown that association of core-SRF with SREfosreduces the flexibility of each partner, suggesting a strong role for dynamics in the adjustment of protein–DNA con-tacts and thereby the specificity of the complex forma-tion [7] Time-resolved fluorescence anisotropy decays

of the modified and native fluoresceinated SREs allow

us to assess differences in dynamics among the three oligonucleotides The results highlight the strong rela-tionships between the base sequence, DNA bending, interactions with water molecules and the internal dynamics in the specific attachment of core-SRF to SREfos

Results

Electric charge distribution in SRE containing oligonucleotides at 10C

The electric charge distribution along the phosphate backbone plays a crucial role in the recognition of DNA by proteins [39] Certain changes within this distribution can affect the fluorescence emission of fluorescein linked to the oligonucleotide [32] In solu-tion at pH 8.5, fluorescein exists as an equilibrium of mono- and dianionic forms Upon excitation at

490 nm, the unlinked fluorescein fluoresces with a maximum at 516 nm The emission spectrum shifts to

520 nm when fluorescein is conjugated to SREfos (Fig 1), the electrostatic potential of DNA generating

a new equilibrium between the mono- and dianionic populations of the fluorescein [7] By contrast, the mutations performed in the native SREfos sequence

do not affect the fluorescein emission profile indica-ting that the fluorescent reporter experiences almost the same environment in SREfos, SREGfos and SREGGfos The electric charges cannot, therefore, be considered responsible for the differences in stoichio-metry observed previously between the complexes

of SREfos, SREGfos and SREGGfos formed with the core-SRF

Interactions between neighboring bases of the SRE oligonucleotides at 10C

The CD spectra of oligonucleotides are influenced by both the base composition of the nearest neighbor and the mutual arrangement of bases [28] The CD spectra of SREfos, SREGfos and SREGGfos, recorded at

10C, are of the B-DNA family with a positive band centered at 272 nm and a negative band close to

250 nm (Fig 2) Slight differences can be assigned to small changes in local interactions introduced by the mutations

Basic character of the temperature effect on SRE

oligonucleotides: singular value decomposition

analysis of Raman spectra

The Raman spectra of the three duplexes are

sensi-tive to temperature variations between 10 and 65C

To find out the basic character of these changes,

each set of spectra was statistically treated by means

of singular value decomposition (SVD) [40] SVD

outputs were similar for the three SREs Those for

SREfos are given in Fig 3 A factor dimension of 3,

means that all Raman spectra obtained between 10

and 65C can be expressed from three spectral

com-ponents (Fig 3) The first component, S1, is an

invariable spectral residuum with an almost constant

V1 contribution in each Raman spectrum The other two components, S2 and S3, account for two types

of change induced by temperature Their contribu-tions, V2 and V3, reveal two kinds of temperature processes separated by a boundary between 30 and

40C: V2 and V3 exhibit an inflexion and a mini-mum While the second dimension (V2, S2) shows spectral features that are common for both transi-tions, the third dimension (V3, S3) reflects the differ-ences between them

Above 40C, spectral changes are related to the melting of the duplexes V2 and V3 show a parallel increase with temperature and the changes induced by temperature are given by the summation of the spec-tral components S2 and S3 The most significant chan-ges include (Fig 3): a decrease in the intensity of the Raman bands of deoxyribose phosphate backbone typical of B-type structures [789, 838, 891 (893),

1092 cm)1] [29,30,41] and of some bands characteris-tic for 2¢-endo ⁄ anti conformation of deoxynucleotides [671 (dT), 681 (dG), 1255 (dA, dC), 1338 cm)1 (dA)] [41–44] By contrast, there is an increase in the Ra-man bands at 729 (dA), 1238 (dT), 1303 (dA), 1488 (dA, dG), and 1667 cm)1 (dT) in response to base destacking in the oligonucleotides [29,30,41–43,45–49] Between 10 and 25C, within the premelting domain, spectral changes are reflected in a gradual increase in the contribution of V2 and a simultaneous decrease in the contribution of V3 V3 is normalized and its amplitude looks very similar for all three duplexes in

a temperature region where the premelting is domin-ant By contrast, V2 is mainly normalized according

to melting and weak variations between the three duplexes can be seen during premelting In this study,

we are interested in the premelting transitions because they reveal subtle variations without dissociation of the DNA strands

Changes occurring in SRE oligonucleotides between 25 and 10C

B-DNA conformation of duplexes at 25C

At 25C, the three duplexes display very similar Raman spectra Several peaks can be assigned to known characteristic vibrational bands (Figs 4–6, upper) Bands from the deoxyribose-phophate back-bone (790, 838, 1093 and 1421 cm)1) at a position diagnostic of the B-type conformation [41,42] are identified together with bands from deoxyoligonucleo-tides [681 (dG), 750 (dT), 1255 (dC) and 1339 cm)1 (dA)] related to the C2¢-endo ⁄ anti conformation [41,42,44] The resemblances between the spectra

-2

0

2

4

Wavelength (nm)

Fig 2 CD spectra of SRE fos (e), SRE Gfos (h) and SRE GGfos (n).

Temperature 10 C Ellipticity is expressed in millidegrees Optical

path length 0.1 cm Oligonucleotide concentration: 10)6M

Wavelength (nm)

Fig 1 Conjugation effect of SRE oligonucleotides on fluorescein

fluorescence emission Fluorescence emission spectrum of

fluo-rescein (—) Fluorescence emission spectrum of fluofluo-rescein

conju-gated to SRE fos (e) Both spectra are normalized The fluorescence

emission spectra of fluorescein conjugated to the three

oligonucleo-tides are identical Spectra obtained at 10 C with an excitation

wavelength at 490 nm.

Fig 3 Results of the factor analysis applied to the set of temperature Raman spectra of SRE fos Raman spectrum Yiat each temperature is decomposed into M independent subspectra S j Upper: (left) Singular values W j evaluating statistical weight of individual spectral compo-nents S j , (right) residual errors for various numbers of considered spectral components M Both panels show that the true factor dimension, i.e the minimum number of spectral components sufficient to approximate all Raman spectra, is 3 Middle: Relevant spectral components

Sj, j ¼ 1, 2, 3 Lower: Coefficients V ij , j ¼ 1, 2, 3, indicating the relative contribution of each spectral component S j into the spectrum Yi Spectral components (S 1 , S 2 , S 3 ) and coefficients (V 1 , V 2 , V 3 ) are normalized so that the sum of their squares over spectral points or tem-perature, respectively, is equal to 1 Dashed lines indicate marker bands of the duplex melting, observable as coincidently oriented peaks in the both S2 and S3 spectral components.

indicate that the three oligonucleotides have very

similar B-DNA conformations

Spectral changes in the three duplexes between 25

and 10C

The effects of decreasing the temperature from 25 to

10C are illustrated by the difference Raman spectra

(Figs 4–6, lower) For the same oligonucleotide, the shape of the difference spectra between two tempera-tures is conserved When we compare the shape of the spectra from one oligonucleotide with the two others, high levels of similarity are also apparent Essentially, the band intensities and a few band positions vary slightly Spectral conservation allows us to make a common analysis of the temperature effect on the three duplexes, in agreement with the similarity of the results provided by their respective SVD analysis Changes of intensity and position of the Raman bands are given

in Table 1

Effect of the temperature decrease on base stacking and backbone geometry

The 790⁄ 784 cm)1 doublet undergoes both an upshift

of its 784 cm)1 component and an increase in the intensity of its 790 cm)1 component, thus expressing changes in the geometry of the phosphodiester group, and⁄ or in the conformation of deoxycytidine or deoxy-thymidine, these becoming closer to the 2¢-endo ⁄ anti geometry [41,42,47] The differential profile around

1339 cm)1 shows that the corresponding adenine band

is upshifted to its position of 2¢-endo ⁄ anti conforma-tion [41] For cytosine, the shift in the 1255 cm)1band

to 1265 cm)1 very probably indicates a change in de-oxynucleoside sugar pucker from the C3¢-endo ⁄ anti family to the C2¢endo ⁄ anti family [41–43] The shift in the 838 cm)1 band toward higher wavenumbers, though moderate, is generally interpreted as a sign of minor groove narrowing [29,41,45] Wavenumber up-shift can be also seen for the sugar vibration at

Fig 4 Raman spectrum of SRE fos at 25 C and the effect of a

decrease in temperature to 10 C Upper: Spectrum at 25 C.

Lower: Temperature effect on the Raman spectrum: spectrum at

10, 15 or 20 C minus spectrum at 25 C The intensity scale is the

same in Figs 4–6.

Fig 5 As Fig 4, but for SRE Gfos

Fig 6 As Fig 4, but for SRE GGfos

Table 1 Temperature-induced change in the Raman spectra of SRE fos , SRE Gfos and SRE GGfos and difference in Raman spectra between SREfosand SREGfosand between SREfosand SREGGfos.

Peak position

at 25 C a,b

Effect of

temperature

decrease

from 25

to 10 C a,c

Difference spectrum

Difference spectrum

3¢-endo ⁄ -anti at 745 [41]

3¢-endo ⁄ anti at 780 [41]

dT 2¢-endo ⁄ anti [41,47]

to minor-groove dimension [41,45]

T CH3rock [45],

dr at 1003 [43]

electrostatic environment [46]

dC [51]

also dG in [51]

dC 2¢-endo ⁄ anti at 1255, shift to 1265 for 3¢-endo ⁄ syn [41] against 2¢-endo ⁄ anti at 1268 [43]; signature of adenine

non Watson–Crick bonding [45]

3¢-endo ⁄ anti at 1335 [41];

dG 2¢-endo ⁄ anti at 1336 [44]

dA, dG [42]

intensity increase in hydrophobic environment of T methyl [29,47]

N7 bonding to guanine causes intensity decrease [49] and frequency downshift [47]

893 cm)1[29] The intensity increase for the 731 (729),

754 (750), 1306 (1303), 1379 (1375), 1490 (1488), 1584

(1577), 1662 (1668) and 1695–1730 cm)1 (dT) bands

results from partial base unstacking affecting mainly

adenine and thymine, and to a lesser degree also

guan-ine [30,41,42,44–49] Globally, Raman bands related to

the sugar–phosphate backbone conformation and to

base-stacking reflect conformational changes taking

place in various regions of the DNA duplexes The

changes induced in our spectra by the decrease in

tem-perature from 25 to 10C are similar to those resulting

from the formation of a sharp bend in the DNA

octamer duplex (HMG box) due to binding of the

human SRY–HMG protein The decrease in

tem-perature results in striking similarities between both

Raman signatures (Figs 4–6, lower) [47,50] We may

therefore conclude that SREfos and its two mutants

exhibit, at 10C, a large population of bent

conform-ers The bend is not limited to the central (A⁄ T)

sequence of the CArG box, but includes the bordering

G⁄ C base pairs, because guanine and cytosine signals

(1488, 1578 cm)1 and 780, 1257, 1299 cm)1,

respect-ively) are also affected [41,42,46,51] The structural

adjustment resulting in a bent population at 10C

underlies a more favored linear B form at higher

tem-peratures The increase in intensity of the 926 (924),

1444 and 1462 cm)1 vibrational bands of deoxyribose

and of the 790 and 1056 cm)1 bands of backbone reflects the disappearance of this linear population [29,30,41,42,45] The increase in both well-resolved bands at 1444 and 1462 cm)1 correlates with a broad band around 1400 cm)1 at 25C of about the same integral intensity, indicating a larger population of lin-ear conformers The upshift and increase in intensity

of the peak at 838 cm)1 suggest that the backbone conformation is altered to the detriment of a more canonical B form [41,45]

Effect of a decrease in temperature on hydrogen-bond interactions and hydration

From 25 to 10C, numerous base vibrations exhibit spectral shifts indicating changes in the hydrogen bond array However, these do not concern regular Watson– Crick hydrogen bonds The upshift of the adenine bands at 1510 cm)1 (sensitive to binding at N7) and

1577 cm)1, like that of the guanine band at 1488 cm)1 (also sensitive to interaction at N7), are signs of hydro-gen-bond formation [42,45–47,49] The downshift of the 1668 cm)1 band to 1662 cm)1 is connected with

a change in hydrogen-bond interaction at the O4 of thymine [41,42,45,48] These changes can be assigned

to a redistribution of water molecules or hydrated ions

on the above-mentioned base This is in accordance with the weak wavenumber downshift of the PO2

Table 1 Continued.

Peak position

at 25 C a,b

Effect of

temperature

decrease

from 25

to 10 C a,c

Difference spectrum

Difference spectrum

dC [43]

extra H-bonding at C ¼ O [41,45]

extra H-bonding at C2 ¼ O [41,45]

› 1736

dG: CO str [44] variable position 1686–1722 [44]

a

Common characteristics for the three DNA duplexes.bPeak positions are in wavenumber units (cm)1) Numbers in bold correspond to well-resolved bands; precision of the peak position ± 1 cm)1 Numbers in standard type correspond to shoulders, asymmetrical or partly overlapped bands, and also to peaks in difference spectra; precision of the peak position ± 3 cm)1 Added are basic characteristics of Raman band intensities: w ¼ weak, m ¼ medium, s ¼ strong, vs ¼ very strong, sh ¼ shoulder, br ¼ broad, as ¼ asymmetric c

Symbols: › inten-sity increase, fl intensity decrease,  upshift of vibrational frequency,  downshift of vibrational frequency If the intensity increase or decrease in the difference spectrum is not pronounced exactly at the frequency corresponding to the basic Raman band position (first col-umn), the position of the peak or nick in the difference spectrum is indicated.dAbbreviations: A, C, G, T ¼ adenine, cytosine, guanine, thy-mine; dA, dC, dG, dT ¼ deoxynucleotide containing given nucleobase; bk ¼ backbone; dr ¼ deoxyribose e In case of overlapping Raman bands of several vibrational modes, the dominating mode is underlined Abbreviations for vibrational modes: str ¼ stretching, def ¼ deforma-tion, breath ¼ breathing, rock ¼ rocking, op ¼ out-of-plane, sym ¼ symmetric.

symmetric stretching vibration (1092 cm)1) expected to

be sensitive to solvent charge interactions in the

envi-ronment of phosphate groups [46]

Effect of mutations

Even though the temperature difference spectra look

similar between one oligonucleotide and the other two

(Figs 4–6, lower), their mutual differences reveal some

disparities These are visible at 10 and 25C in the

spectra shown in Figs 7 and 8, respectively, and in

Table 1 At a given temperature, spectra of SREfosand

SREGfos bearing one mutation are very similar, but

they differ much more significantly from the spectrum

of SREGGfosbearing two mutations

Effect on G:C base pairs

As expected, the mutations entail visible, local

con-formational changes between the native C)5C)4 and

single mutated C)5G)4steps (SREGfos), and the double

mutated G)5G)4 step (SREGGfos) The main effect of

the mutations concerns the region of the two G:C base

pairs, whose orientation is reversed There are signs

of increased intensity for several guanosine signals

(troughs at 679, 1321, 1361, 1488 and 1578 cm)1)

[41–44,46,48], including the markers of deoxyguanosine

2¢-endo ⁄ anti conformation (679 and 1361 cm)1) and

also the 1321 cm)1 band considered to be a 2¢-endo ⁄

syn conformation marker [44]; the increased intensity

of several of these bands reflects increased unstacking

of the guanine residue By contrast, several positive peaks in the difference spectra (780, 1257 and

1299 cm)1) are attributable to a decreased cytidine intensity [41,42,51] They indicate that, in the case of cytidine, the mutation causes better stacking and also reduces the probability of the 3¢-endo ⁄ anti conforma-tion (the 780 cm)1band) [41]

In the spectral differences at 10 and 25C the muta-tional effects are conserved for the guanosine bands, whereas they are substantially weaker at increased tem-perature for the cytidine bands

Effect on hydrogen-bond interactions, hydration and stability of the various SREs

At 10 C (Fig 7), the negative band at 755 cm)1 attributed to the deoxythymidine 2¢-endo ⁄ anti confor-mation appears somewhat more pronounced in the mutated versions [41] The two deoxyribose vibration bands (positive peaks at 885 and 928 cm)1) become less intensive in both mutant spectra [29,30,45] For the double mutant SREGGfos, the simultaneous upshift

of the 1668 cm)1 band suggests a weakening of the extra hydrogen bonding of the thymine carbonyl with the surrounding water molecules [41,42,45,48] Because

no bands appear around 1093 cm)1 the electrostatic environment of the three duplexes cannot be distin-guished [46]

At 25C (Fig 8), the difference in the Raman spec-tra between the oligonucleotides increases The differ-ent intensities of the bands at 1402 cm)1 and at 790,

838, 927, 1056 cm)1 of the deoxyribose and the back-bone [29,30,41,45] reflect the relative disappearance of

Fig 7 Difference in Raman spectra at 10 C between SRE fos

and SRE Gfos and between SRE fos and SRE GGfos The intensity scale is

the same as in Figs 4–6.

Fig 8 As Fig 7, but at 25 C.

the bend population for the benefit of the linear one.

Concurrently, the bands from extra hydrogen bonding

at thymine (1662, 1699 cm)1) [41,42,45,48] amplified

by the increase in temperature, varies with the band at

1093 cm)1 (positive peak at 1086 cm)1, trough at

1098 cm)1), most probably due to modified

interac-tions between the (A⁄ T) domain and solvent

mole-cules These changes concern mainly the SREGGfosand

to a lesser extent the SREGfos Thus, an increase in

temperature decreases the thermal stability of the bent

form in the order: SREfos< SREGfos< SREGGfos

The bent structure of SREfosis the most stable and

preserved of the three duplexes, whereas the double

mutation brings about a higher instability of that

structure

Internal dynamics of SRE helices

The dynamics of the three SRE oligonucleotides were

assessed using time-resolved fluorescence anisotropy

decays with the fluorescein group fixed at the 5¢-end as

a fluorescence reporter During the lifetime of its

exci-ted state (4 ns), the fluorescein group is involved in

several motions: rotation as a whole, together with the

internal motions of the oligonucleotide; and the proper

rotations of the fluorophore around its link with the

oligonucleotide Correlation times for the

multiexpo-nential anisotropy decays with their relative

propor-tions are shown in Table 2 The shortest correlation

time (i.e.F ¼ 0.4 ns) carries the strongest weight in

the composite decay This correlation time is linked

to the time of fluorescein rotation around its link with

the oligonucleotide The correlation time for rotation

of the SRE molecule as a whole, estimated to be 10 ns

from hydrodynamic measurements [7,52], was hard to

detect in our experiments In any case, the fast depo-larization process due to fluorescein motions prevents monitoring of the entire oligonucleotide rotation Because the fluorescent reporter experiences the same environment for the three oligonucleotides, we con-clude that the longest correlation time reflects the internal dynamics of helix strands that drive fluoresc-ein with them The longest correlation time for SREfos, i.e F ¼ 3.2 ns, slows to F ¼ 3.9 ns in SREGfos, whereas the double mutation shortens it to F ¼ 1.8 ns

in SREGGfos The inverse of the correlation time (1⁄ F) represents the twisting oscillation frequency (m) of the double helix The oscillation frequency increases in the order (Table 2): SREGfos < SREfos< SREGGfos Table 2 also gives the statistical weight (b) for the lon-gest correlation times which increases in the order: SREfos< SREGfos< SREGGfos

For each oligonucleotide, this weight decreases when the temperature increases from 10 to 30C (not shown), indicating a lower population that depolarizes

at higher temperature Because the population of the bent form decreases at higher temperature, we must assume that the linear form does not give a detectable depolarization signal Thus, fluorescence anisotropy decay mainly detects the helix twisting of the bent form offering enough thermal amplitude motions In addition, b-value and thermal instability of the bent form detected using differences in Raman spectra between the oligonucleotides increase in the same order

Discussion

The Cfi G mutations at the )5 and )4 positions of the CArG box alter the binding stoichiometry in a dra-matic manner [7] Here we show that, at 10C, such mutations do not affect electric charge repartition along the oligonucleotides and preserve the same B-DNA conformation Essentially, the interactions at the mutated positions are modified together with the arrangement of water molecules and the internal dynamics

Premelting effect on the equilibrium of the bent linear form

The premelting transition has been studied in detail by Raman spectroscopy for alternating [poly(dA–dT)]2 and homogenous poly(dA):poly(dT) sequences [30,45] The similarity to the effects of temperature on our Raman spectra emphasizes its influence on the six cen-tral (A⁄ T) base pairs of the CArG boxes Detailed analysis of Raman spectra has confirmed that the

Table 2 Relation between parameters of the fluorescence

aniso-tropy decays of fluorescein labeling the various SRE

oligonucleo-tides and the number of bound core-SRF monomers at 10 C.

F a

ns

(± 0.1 ns)

b b

% (± 2%)

m ¼ 1 U c

a F, correlation time The longest correlation time characterizes the

internal motion of the DNA duplex b b, weight of the exponential

component. c m, oscillation frequency. d N, number of core-SRF

monomer bound to DNA fragment [7].

premelting transition conserves the basic local

confor-mation features of B-DNA (A⁄ T)-rich sequences have

been found to be highly polymorphic and depend

strongly on the temperature [53,54] Indeed, the change

in the array of the hydrogen bonds at thymine of

SREGGfos is probably a sign of perturbation in the

hydration scheme along the minor groove of the (A⁄ T)

domain The G–C base pair is characterized by a large

dipole and both inversions change the local electric

charge repartition at)5 and )4 positions of the CArG

box, and as a consequence the interactions with water

molecules of the (A⁄ T) domain [55,56] Premelting

transitions are ascribed to the disruption of water

molecules specifically bound to DNA [31,45,57] The

presence of a ‘low-temperature form’, referred to as

B¢-type DNA, is correlated with tight binding between

water molecules and bases, especially in the narrow

minor groove of the (A⁄ T) domains [53,54,58]

At low temperatures, between 5 and 10C, free

SREfos appears more bent using Raman spectroscopy

than was found using electrophoresis [23,59] Relevant

to the vibrational timescale (10)14 s), Raman

spectros-copy allows the signals of the bent and linear

conform-ers to be differentiated whatever their convconform-ersion time,

whereas electrophoretic techniques average the signals

of both conformers [59] Thus, it is more a transient

bent population than a stable one that is observed in

solution From one oligonucleotide to the other two,

the temperature difference Raman spectra (Figs 4–6),

like the difference spectra at 10 and 25C (Figs 7,8),

exhibit a high degree of spectral pattern conservation

with uniform low-intensity variations The SREfosand

its two mutants oscillate between a bent and a linear

form keeping the same average conformations Thus,

an increase in temperature displaces the equilibrium,

increasing the amplitude of motion around the regular

states within the frame of the same average geometries

These results suggest that the conversion process arises

from global thermal fluctuations of the

oligonucleo-tides and the mutations mainly influence the

probabil-ity of their occurrence [60]

Bending magnitude of SREfos

In order to evaluate the bend angle induced by the

decrease in temperature from 25 to 10C, the Raman

spectral changes for SREfoswere compared with those

resulting from the formation of a sharp bend in a

DNA octamer duplex (HMG box) upon interaction

with the SRY(HMG) protein [47] The CArG and

HMG boxes have very similar proportions of A:T vs

G:C base pairs (6:4 in our case and 5:3 in HMG box),

and approximately the same size region is expected to

be subject to a sharp bend Moreover, the SREs used

in this study (20-mers) contain 2.5 times more nucleo-tides than the HMG box (octamer used for compar-ison) The spectral changes occurring in SREfos between 10 and 25C correspond to approximately half of that caused by the SRY–HMG protein in the HMG box Otherwise, the temperature-induced struc-tural changes in the Raman spectra during premelting are mainly characterized, in SVD analysis, by variation

in the V2 contribution of the spectral component S2 Actually, the temperature profile of the V2 contribu-tion is in accordance with the reduccontribu-tion in the bent population in the oligonucleotide Thereby, we can deduce that 10C corresponds closely to the tempera-ture transition between the bent form and the linear form, since their populations are roughly equivalent The agreement between our results and those reported

by Benevides et al [47] for the 70 sharp bend induced

in the HMG box seems very interesting Indeed, the bend determined by Raman for the free SREfos in solution is roughly similar to that formed in SREfos

in the crystal of its complex with the core-SRF [24] This study does not provide information on the local repartition of the angles involved in the SREfos helix bending

Relative effect of bending strain There are several indications of a redistribution of the strains exerted on the oligonucleotide by the bend: partial unstacking of some adenine, thymine and guanine bases and a more distinct presence of 2¢-endo conformations of furanose rings at 10 C against a higher percentage of 3¢-endo ⁄ anti at 25 C Because the bend is present at low temperature, its stabilization must be favored from the point of view

of enthalpy, but unfavored from the point of view of entropy A 25C, the higher entropy of the linear form is likely due to its higher flexibility, the higher mobility of the hydration shell, or both In the curved conformation, the strain exerted on the secon-dary structure of the double helix increases its tor-sional stiffness [61]

Dynamic effects of mutations on SRE helices

Gfi C base mutations at positions)4 and )5 of the CArG box induce only slight local structural differ-ences but important interactional changes between the bases The extensive empirical study of El Hassan and Calladine [56] showed that the CA step adopts a wide continuous range of conformations However, the persistence of the backbone conformation restricts