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

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CfiG base mutations in the CArG box of c-fos serumresponse element alter its bending flexibilityConsequences for core-SRF recognitionJosef Stepanek1,2,*, Michel Vincent3,, Pierre-Yves Turpin1, Denise Paulin2, Serge Fermandjian4,5,Bernard Alpert2and Christian Zentz11 Laboratoire de Biophysique Mole´culaire Cellulaire & Tissulaire, Universite´Pierre et Marie Curie, Evry, France2 Laboratoire de Biologie Mole´culaire de la Diffe´renciation, Universite´Denis Diderot, Paris, France3 LURE, Universite´Paris-Sud, Orsay, France4De´partement de Biologie et Pharmacologie Structurales, Ecole Normale Supe´rieure de Cachan, France5 Institut Gustave Roussy, Villejuif, FranceSpecific binding of the serum response factor (SRF) tothe serum response element (SRE) requires a consensussequence CC(A ⁄ T)6GG, the CArG box [1–7]. Thetranscriptional 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-affinityCArG box, whereas many muscle-specific genescontain two or more CArG boxes. However, thesecarry substitutions with G or C nucleotides withintheir (A ⁄ T) domain, thus lowering the affinity [15–19].KeywordsCArG box; c-fos; DNA bending; DNAdynamics; serum response elementCorrespondenceC. Zentz, Laboratoire de BiophysiqueMole´culaire Cellulaire & Tissulaire,Universite´Pierre et Marie Curie, CNRSUMR 7033, GENOPOLE Campus 1, 5 rueHenri Desbrue`res, 91030 Evry Cedex,FranceFax: +33 1 69 87 43 60Tel: +33 1 69 87 43 52E-mail: zentz@ccr.jussieu.fr*Permanent addressCharles University, Faculty of Mathematicsand Physics, Prague, Czech RepublicPresent addressIBBMC, Universite´Paris-Sud, Orsay France(Received 22 December 2006, revised 20February 2007, accepted 2 March 2007)doi:10.1111/j.1742-4658.2007.05768.xBy binding to the CArG box sequence, the serum response factor (SRF)activates several muscle-specific genes, as well as genes that respond tomitogens. The core domain of the SRF (core-SRF) binds as a dimer to theCArG box C)5C)4A)3T)2A)1T+1T+2A+3G+4G+5of the c- fos serumresponse element (SREfos). However, previous studies using 20-mer DNAshave shown that the binding stoichiometry of core-SRF is significantlyaltered 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, PValat, 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, SREGfosand SREGGfosin aqueoussolution. Their CD spectra were of the B-DNA type with small differencesgenerated by variations in the mutual arrangement of the base pairs. Ana-lysis by singular value decomposition of a set of Raman spectra recordedas a function of temperature, revealed a premelting transition associatedwith a conformational shift in the DNA double helices from a bent to alinear form. Time-resolved fluorescence anisotropy shows that the fluores-cein reporter linked to the oligonucleotide 5¢-ends experiences twistingmotions of the double helices related to the interconversion between bentand linear conformers. The three SREs present various bent populationssubmitted, however, to particular internal dynamics, decisive forthe mutual adjustment of binding partners and therefore specific complexformation.Abbreviationscore-SRF, core domain of the serum response factor; SRE, serum response element; SRF, serum response factor; SVD, singular valuedecomposition.FEBS Journal 274 (2007) 2333–2348 ª 2007 The Authors Journal compilation ª 2007 FEBS 2333Strong-affinity CArG boxes are likely to bind SRFconstitutively and genes appear to be regulated primar-ily during the post-SRF binding step, owing to interac-tions with accessory proteins [20]. Weaker affinityCArG boxes may offer additional control through amechanism that influences SRF binding, i.e. by mutualcombined interactions of CArG boxes and accessoryproteins [21,22].The core domain of the SRF (core-SRF) binds tothe CArG box as a homodimer [7,23,24]. The specificcore-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 SRFcauses 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 theCArG box sequence [22]. To understand the originof these effects this study focuses on the three 20-meroligonucleotides: SREfos, SREGfosand SREGGfos. TheSREfossequence, 5¢-d(GGATGTC)5C)4A)3T)2A)1T+1T+2A+3G+4G+5ACAT)-3¢, embodies the nativeCArG box of the c-fos enhancer (CArG box numbered)[2,27], whereas SREGfoscarries the single C)5fi Gmutation and SREGGfosthe double C)5C)4fi GGmutation within their CArG box. A previous report hasshown that the parent SREfosbound a core-SRFhomodimer, whereas the single mutant SREGfosand thedouble mutant SREGGfosbound one and four mono-mers (on average), respectively [7]. This highlights therole of the base sequence at the border of the A ⁄ T trackin the specific complex assembly and functional organ-ization. How mutations affect binding of the core-SRFand generate a lack of defined stoichiometry is an openquestion. Thus, we carried out a comparative analysis ofthe three oligonucleotides using Raman, CD and fluor-escence spectroscopies in order to detect their mutualstructural, electrostatic and dynamical differences. CDand Raman are sensitive to small structural changes[28,29]. In addition, Raman scattering is a powerfulmeans of clearing up the various sensitivities of thenucleic acid chains to temperature [30,31]. Fluorescencestudies require a fluorophore reporter, such as fluo-rescein, chemically fixed to the oligonucleotides. Thefluorescein fluorescence signal arises from the overlap-ping emissions of the mono- and dianionic protolyticstates [32], which are sensitive to the electric chargedistribution on the DNA. Chain DNA dynamics havebeen extensively studied [33–37]. DNA is intrinsicallyflexible, but this flexibility varies from one DNAto another [38]. To date, little is known about therelationships between the ability of DNA to bend andits effects on protein binding. Previous studies haveshown that association of core-SRF with SREfosreducesthe flexibility of each partner, suggesting a strong rolefor dynamics in the adjustment of protein–DNA con-tacts and thereby the specificity of the complex forma-tion [7]. Time-resolved fluorescence anisotropy decaysof the modified and native fluoresceinated SREs allowus to assess differences in dynamics among the threeoligonucleotides. The results highlight the strong rela-tionships between the base sequence, DNA bending,interactions with water molecules and the internaldynamics in the specific attachment of core-SRF toSREfos.ResultsElectric charge distribution in SRE containingoligonucleotides at 10°CThe electric charge distribution along the phosphatebackbone plays a crucial role in the recognition ofDNA by proteins [39]. Certain changes within thisdistribution can affect the fluorescence emission offluorescein linked to the oligonucleotide [32]. In solu-tion at pH 8.5, fluorescein exists as an equilibrium ofmono- and dianionic forms. Upon excitation at490 nm, the unlinked fluorescein fluoresces with amaximum at 516 nm. The emission spectrum shifts to520 nm when fluorescein is conjugated to SREfos(Fig. 1), the electrostatic potential of DNA generatinga new equilibrium between the mono- and dianionicpopulations of the fluorescein [7]. By contrast, themutations performed in the native SREfossequencedo not affect the fluorescein emission profile indica-ting that the fluorescent reporter experiences almostthe same environment in SREfos, SREGfosandSREGGfos. The electric charges cannot, therefore, beconsidered responsible for the differences in stoichio-metry observed previously between the complexesof SREfos, SREGfosand SREGGfosformed with thecore-SRF.Interactions between neighboring bases of theSRE oligonucleotides at 10°CThe CD spectra of oligonucleotides are influenced byboth the base composition of the nearest neighborand the mutual arrangement of bases [28]. The CDspectra of SREfos, SREGfosand SREGGfos, recorded at10 °C, are of the B-DNA family with a positive bandcentered at 272 nm and a negative band close to250 nm (Fig. 2). Slight differences can be assigned tosmall changes in local interactions introduced by themutations.Implication of CArG sequence in SRE flexibility J. Stepanek et al.2334 FEBS Journal 274 (2007) 2333–2348 ª 2007 The Authors Journal compilation ª 2007 FEBSBasic character of the temperature effect on SREoligonucleotides: singular value decompositionanalysis of Raman spectraThe Raman spectra of the three duplexes are sensi-tive to temperature variations between 10 and 65 °C.To find out the basic character of these changes,each set of spectra was statistically treated by meansof singular value decomposition (SVD) [40]. SVDoutputs were similar for the three SREs. Those forSREfosare given in Fig. 3. A factor dimension of 3,means that all Raman spectra obtained between 10and 65 °C can be expressed from three spectral com-ponents (Fig. 3). The first component, S1,isaninvariable spectral residuum with an almost constantV1contribution in each Raman spectrum. The othertwo components, S2and S3, account for two typesof change induced by temperature. Their contribu-tions, V2and V3, reveal two kinds of temperatureprocesses separated by a boundary between 30 and40 °C: V2and V3exhibit an inflexion and a mini-mum. While the second dimension (V2,S2) showsspectral features that are common for both transi-tions, the third dimension (V3,S3) reflects the differ-ences between them.Above 40 °C, spectral changes are related to themelting of the duplexes. V2and V3show a parallelincrease with temperature and the changes induced bytemperature are given by the summation of the spec-tral components S2and S3. The most significant chan-ges include (Fig. 3): a decrease in the intensity of theRaman bands of deoxyribose phosphate backbonetypical 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 basedestacking in the oligonucleotides [29,30,41–43,45–49].Between 10 and 25 °C, within the premelting domain,spectral changes are reflected in a gradual increase inthe contribution of V2and a simultaneous decrease inthe contribution of V3.V3is normalized and itsamplitude looks very similar for all three duplexes ina temperature region where the premelting is domin-ant. By contrast, V2is mainly normalized accordingto melting and weak variations between the threeduplexes can be seen during premelting. In this study,we are interested in the premelting transitions becausethey reveal subtle variations without dissociation ofthe DNA strands.Changes occurring in SRE oligonucleotidesbetween 25 and 10°CB-DNA conformation of duplexes at 25°CAt 25 °C, the three duplexes display very similarRaman spectra. Several peaks can be assigned toknown characteristic vibrational bands (Figs 4–6,upper). Bands from the deoxyribose-phophate back-bone (790, 838, 1093 and 1421 cm)1) at a positiondiagnostic of the B-type conformation [41,42] areidentified 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-2024200 240 280Wavelength (nm)Ellipticity (m.deg.)Fig. 2. CD spectra of SREfos(e), SREGfos(h) and SREGGfos(n).Temperature 10 °C. Ellipticity is expressed in millidegrees. Opticalpath length 0.1 cm. Oligonucleotide concentration: 10)6M.500 520 540 560 580Wavelength (nm)Fluorescence intensity (a.u.)Fig. 1. Conjugation effect of SRE oligonucleotides on fluoresceinfluorescence emission. Fluorescence emission spectrum of fluo-rescein (—). Fluorescence emission spectrum of fluorescein conju-gated to SREfos(e). Both spectra are normalized. The fluorescenceemission spectra of fluorescein conjugated to the three oligonucleo-tides are identical. Spectra obtained at 10 °C with an excitationwavelength at 490 nm.J. Stepanek et al. Implication of CArG sequence in SRE flexibilityFEBS Journal 274 (2007) 2333–2348 ª 2007 The Authors Journal compilation ª 2007 FEBS 2335Fig. 3. Results of the factor analysis applied to the set of temperature Raman spectra of SREfos. Raman spectrum Yiat each temperature isdecomposed into M independent subspectra Sj. Upper: (left) Singular values Wjevaluating statistical weight of individual spectral compo-nents Sj, (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 componentsSj,j¼ 1, 2, 3. Lower: Coefficients Vij,j¼ 1, 2, 3, indicating the relative contribution of each spectral component Sjinto the spectrum Yi.Spectral components (S1,S2,S3) and coefficients (V1,V2,V3) 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 inthe both S2 and S3 spectral components.Implication of CArG sequence in SRE flexibility J. Stepanek et al.2336 FEBS Journal 274 (2007) 2333–2348 ª 2007 The Authors Journal compilation ª 2007 FEBSindicate that the three oligonucleotides have verysimilar B-DNA conformations.Spectral changes in the three duplexes between 25and 10°CThe effects of decreasing the temperature from 25 to10 °C are illustrated by the difference Raman spectra(Figs 4–6, lower). For the same oligonucleotide, theshape of the difference spectra between two tempera-tures is conserved. When we compare the shape of thespectra from one oligonucleotide with the two others,high levels of similarity are also apparent. Essentially,the band intensities and a few band positions varyslightly. Spectral conservation allows us to make acommon analysis of the temperature effect on the threeduplexes, in agreement with the similarity of the resultsprovided by their respective SVD analysis. Changes ofintensity and position of the Raman bands are givenin Table 1.Effect of the temperature decrease on base stackingand backbone geometryThe 790 ⁄ 784 cm)1doublet undergoes both an upshiftof its 784 cm)1component and an increase in theintensity of its 790 cm)1component, thus expressingchanges in the geometry of the phosphodiester group,and ⁄ or in the conformation of deoxycytidine or deoxy-thymidine, these becoming closer to the 2¢-endo ⁄ antigeometry [41,42,47]. The differential profile around1339 cm)1shows that the corresponding adenine bandis upshifted to its position of 2¢-endo ⁄ anti conforma-tion [41]. For cytosine, the shift in the 1255 cm)1bandto 1265 cm)1very probably indicates a change in de-oxynucleoside sugar pucker from the C3¢-endo ⁄ antifamily to the C2¢endo ⁄ anti family [41–43]. The shift inthe 838 cm)1band toward higher wavenumbers,though moderate, is generally interpreted as a sign ofminor groove narrowing [29,41,45]. Wavenumber up-shift can be also seen for the sugar vibration atFig. 4. Raman spectrum of SREfosat 25 °C and the effect of adecrease in temperature to 10 °C. Upper: Spectrum at 25 °C.Lower: Temperature effect on the Raman spectrum: spectrum at10, 15 or 20 °C minus spectrum at 25 °C. The intensity scale is thesame in Figs 4–6.Fig. 5. As Fig. 4, but for SREGfos.Fig. 6. As Fig. 4, but for SREGGfos.J. Stepanek et al. Implication of CArG sequence in SRE flexibilityFEBS Journal 274 (2007) 2333–2348 ª 2007 The Authors Journal compilation ª 2007 FEBS 2337Table 1. Temperature-induced change in the Raman spectra of SREfos, SREGfosand SREGGfosand difference in Raman spectra betweenSREfosand SREGfosand between SREfosand SREGGfos.Peak positionat 25 °Ca,bEffect oftemperaturedecreasefrom 25to 10 °Ca,cDifferencespectrumSREfos-SREGfos cDifferencespectrumSREfos-SREGGfos cAssignmentd,eSignificanced10 °C25°C10°C25°C671 m fl 662dT, dA [41,42] dT 2¢-endo ⁄ anti at 665 [41]681 m › 691 flflfl fldG [41,42] dG 2¢-endo ⁄ anti at 684 [41,44]729 s › 731 A breath [41,42] hypochromic [45,49]750 m › 754 fl 755 fl 755 dT [41] dT 2¢-endo ⁄ anti at 748,3¢-endo ⁄ -anti at 745 [41]784 m, sh Þ›780 › 780 › 780 › 780 dC [41,42] dC 2¢-endo ⁄ anti at 782,3¢-endo ⁄ anti at 780 [41]790 vs ›››bk O-P-O str + dr, dT [41] B-DNA g––g–of a ⁄ f torsiondT 2¢-endo ⁄ anti [41,47]838 m ›Þ › bk O-P-O str [41] B-DNA, exact position sensitiveto minor-groove dimension [41,45]893 w ÞÜ›885 Ü dr C2¢H2rock [29,30] B DNA, sensitive to premelting [30]924 w, br, as ››928 › 926 › 928 › 928 dr ring str [45] sensitive to B-B¢ transition [30]973 w, br › T C6H op-def [45], bk [42]1013 m, br ››1006 › 1006 › 1006 › 1005 G NH def [44],TCH3rock [45],dr at 1003 [43]1057 w, as ›››bk C-O str [29,30]1093 vs ÜÜPO2–sym str [41] B-DNA [41], sensitive toelectrostatic environment [46]1144 w › 1149 dT [42,45]1178 w, as flfl1186 dT [42,45]; dG [29,43],dC [51]1213 w, br, sh ››1218 › 1218 dT, dA [41,42]; dG [43] dT 2¢-endo ⁄ anti at 1208 [41]1240 w, sh fl dT [42], dC [43] T hypochromic [47]1255 s › 1265–1269 › 1258 › 1257 › 1270 dC, dA, dT [42],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 adeninenon Watson–Crick bonding [45]1294 m,sh › 1299 › 1298 › 1299 › 1299 dC [42,51]1303 s ›dA, dT [42,45] dA hypochromic [30]fl 1321 fl 1321 fl 1322 dG [43,44] dG 2¢-endo ⁄ syn [44])1339 vs, br ÞdA, dG [41,42] dA 2¢-endo ⁄ anti at 1339,3¢-endo ⁄ anti at 1335 [41];dG 2¢-endo ⁄ anti at 1336 [44]fl 1361 fl 1363 fl 1361 fl 1364 dG [44] dG 2¢-endo ⁄ anti [44]1375 vs › 1379TCH3def [45];dA, dG [42]intensity increase in hydrophobicenvironment of T methyl [29,47]fl 1396–1403 fl 14021421 sdrC5¢H2def, dA [42] B-DNA [41]1444 w, br › dr C5¢H2def [42]1462 w › dr C2¢H2def [42]1488 vs, as ›Þ fl fl fl flG imi ring [46], dA, dT [42] hypochromic [41,47];N7 bonding to guanine causesintensity decrease [49] andfrequency downshift [47]1510 m ›Þ fldA, dC [42] upshift with A N7 bonding [45]1532 w, sh dC, dG [42]1577 vs ›Þ fl1580 fl 1583 flfl1574 dG, dA [42] G, A hypochromic [41,45]Implication of CArG sequence in SRE flexibility J. Stepanek et al.2338 FEBS Journal 274 (2007) 2333–2348 ª 2007 The Authors Journal compilation ª 2007 FEBS893 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) bandsresults from partial base unstacking affecting mainlyadenine and thymine, and to a lesser degree also guan-ine [30,41,42,44–49]. Globally, Raman bands related tothe sugar–phosphate backbone conformation and tobase-stacking reflect conformational changes takingplace in various regions of the DNA duplexes. Thechanges induced in our spectra by the decrease in tem-perature from 25 to 10 °C are similar to those resultingfrom the formation of a sharp bend in the DNAoctamer duplex (HMG box) due to binding of thehuman SRY–HMG protein. The decrease in tem-perature results in striking similarities between bothRaman signatures (Figs 4–6, lower) [47,50]. We maytherefore conclude that SREfosand its two mutantsexhibit, at 10 °C, 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 borderingG ⁄ C base pairs, because guanine and cytosine signals(1488, 1578 cm)1and 780, 1257, 1299 cm)1, respect-ively) are also affected [41,42,46,51]. The structuraladjustment resulting in a bent population at 10 °Cunderlies a more favored linear B form at higher tem-peratures. The increase in intensity of the 926 (924),1444 and 1462 cm)1vibrational bands of deoxyriboseand of the 790 and 1056 cm)1bands of backbonereflects the disappearance of this linear population[29,30,41,42,45]. The increase in both well-resolvedbands at 1444 and 1462 cm)1correlates with a broadband around 1400 cm)1at 25 °C of about the sameintegral intensity, indicating a larger population of lin-ear conformers. The upshift and increase in intensityof the peak at 838 cm)1suggest that the backboneconformation is altered to the detriment of a morecanonical B form [41,45].Effect of a decrease in temperature on hydrogen-bondinteractions and hydrationFrom 25 to 10 °C, numerous base vibrations exhibitspectral shifts indicating changes in the hydrogen bondarray. However, these do not concern regular Watson–Crick hydrogen bonds. The upshift of the adeninebands at 1510 cm)1(sensitive to binding at N7) and1577 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 ofthe 1668 cm)1band to 1662 cm)1is connected witha change in hydrogen-bond interaction at the O4 ofthymine [41,42,45,48]. These changes can be assignedto a redistribution of water molecules or hydrated ionson the above-mentioned base. This is in accordancewith the weak wavenumber downshift of the PO2–Table 1. Continued.Peak positionat 25 °Ca,bEffect oftemperaturedecreasefrom 25to 10 °Ca,cDifferencespectrumSREfos-SREGfos cDifferencespectrumSREfos-SREGGfos cAssignmentd,eSignificanced10 °C25°C10°C25°C1602 w, sh dC [42], G N1H def [41]1652 m, sh T (C4O ⁄ C5C6) str [42],dC [43]1668 s, br › 1662 Ü›1662 T (C4O ⁄ C5C6) str [42,48] shift to 1662 in case ofextra H-bonding at C ¼ O [41,45]› 1695–1720 › 1699 T C2O str [42,48] at 1689, shift to 1681 in case ofextra H-bonding at C2 ¼ O [41,45]› 1730 › 1711› 1736dG: CO str [44] variable position 1686–1722 [44]aCommon characteristics for the three DNA duplexes.bPeak positions are in wavenumber units (cm)1). Numbers in bold correspond towell-resolved bands; precision of the peak position ± 1 cm)1. Numbers in standard type correspond to shoulders, asymmetrical or partlyoverlapped bands, and also to peaks in difference spectra; precision of the peak position ± 3 cm)1. Added are basic characteristics of Ramanband intensities: w ¼ weak, m ¼ medium, s ¼ strong, vs ¼ very strong, sh ¼ shoulder, br ¼ broad, as ¼ asymmetric.cSymbols: › inten-sity increase, fl intensity decrease, Þ upshift of vibrational frequency, Ü downshift of vibrational frequency. If the intensity increase ordecrease 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.eIn case of overlapping Ramanbands 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.J. Stepanek et al. Implication of CArG sequence in SRE flexibilityFEBS Journal 274 (2007) 2333–2348 ª 2007 The Authors Journal compilation ª 2007 FEBS 2339symmetric stretching vibration (1092 cm)1) expected tobe sensitive to solvent charge interactions in the envi-ronment of phosphate groups [46].Effect of mutationsEven though the temperature difference spectra looksimilar between one oligonucleotide and the other two(Figs 4–6, lower), their mutual differences reveal somedisparities. These are visible at 10 and 25 °C in thespectra shown in Figs 7 and 8, respectively, and inTable 1. At a given temperature, spectra of SREfosandSREGfosbearing one mutation are very similar, butthey differ much more significantly from the spectrumof SREGGfosbearing two mutations.Effect on G:C base pairsAs expected, the mutations entail visible, local con-formational changes between the native C)5C)4andsingle mutated C)5G)4steps (SREGfos), and the doublemutated G)5G)4step (SREGGfos). The main effect ofthe mutations concerns the region of the two G:C basepairs, whose orientation is reversed. There are signsof increased intensity for several guanosine signals(troughs at 679, 1321, 1361, 1488 and 1578 cm)1)[41–44,46,48], including the markers of deoxyguanosine2¢-endo ⁄ anti conformation (679 and 1361 cm)1) andalso the 1321 cm)1band considered to be a 2¢-endo ⁄syn conformation marker [44]; the increased intensityof several of these bands reflects increased unstackingof the guanine residue. By contrast, several positivepeaks in the difference spectra (780, 1257 and1299 cm)1) are attributable to a decreased cytidineintensity [41,42,51]. They indicate that, in the case ofcytidine, the mutation causes better stacking and alsoreduces the probability of the 3¢-endo ⁄ anti conforma-tion (the 780 cm)1band) [41].In the spectral differences at 10 and 25 °C 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, hydrationand stability of the various SREsAt 10 °C (Fig. 7), the negative band at 755 cm)1attributed to the deoxythymidine 2¢-endo ⁄ anti confor-mation appears somewhat more pronounced in themutated versions [41]. The two deoxyribose vibrationbands (positive peaks at 885 and 928 cm)1) becomeless intensive in both mutant spectra [29,30,45]. Forthe double mutant SREGGfos, the simultaneous upshiftof the 1668 cm)1band suggests a weakening of theextra hydrogen bonding of the thymine carbonyl withthe surrounding water molecules [41,42,45,48]. Becauseno bands appear around 1093 cm)1the electrostaticenvironment of the three duplexes cannot be distin-guished [46].At 25 °C (Fig. 8), the difference in the Raman spec-tra between the oligonucleotides increases. The differ-ent intensities of the bands at 1402 cm)1and at 790,838, 927, 1056 cm)1of the deoxyribose and the back-bone [29,30,41,45] reflect the relative disappearance ofFig. 7. Difference in Raman spectra at 10 °C between SREfosandSREGfosand between SREfosand SREGGfos. The intensity scale isthe same as in Figs 4–6.Fig. 8. As Fig. 7, but at 25 °C.Implication of CArG sequence in SRE flexibility J. Stepanek et al.2340 FEBS Journal 274 (2007) 2333–2348 ª 2007 The Authors Journal compilation ª 2007 FEBSthe bend population for the benefit of the linear one.Concurrently, the bands from extra hydrogen bondingat thymine (1662, 1699 cm)1) [41,42,45,48] amplifiedby the increase in temperature, varies with the band at1093 cm)1(positive peak at 1086 cm)1, trough at1098 cm)1), most probably due to modified interac-tions between the (A ⁄ T) domain and solvent mole-cules. These changes concern mainly the SREGGfosandto a lesser extent the SREGfos. Thus, an increase intemperature decreases the thermal stability of the bentform in the order: SREfos< SREGfos< SREGGfos.The bent structure of SREfosis the most stable andpreserved of the three duplexes, whereas the doublemutation brings about a higher instability of thatstructure.Internal dynamics of SRE helicesThe dynamics of the three SRE oligonucleotides wereassessed using time-resolved fluorescence anisotropydecays with the fluorescein group fixed at the 5¢-end asa fluorescence reporter. During the lifetime of its exci-ted state (4 ns), the fluorescein group is involved inseveral motions: rotation as a whole, together with theinternal motions of the oligonucleotide; and the properrotations of the fluorophore around its link with theoligonucleotide. Correlation times for the multiexpo-nential anisotropy decays with their relative propor-tions are shown in Table 2. The shortest correlationtime (i.e. F ¼ 0.4 ns) carries the strongest weight inthe composite decay. This correlation time is linkedto the time of fluorescein rotation around its link withthe oligonucleotide. The correlation time for rotationof the SRE molecule as a whole, estimated to be 10 nsfrom hydrodynamic measurements [7,52], was hard todetect in our experiments. In any case, the fast depo-larization process due to fluorescein motions preventsmonitoring of the entire oligonucleotide rotation.Because the fluorescent reporter experiences the sameenvironment for the three oligonucleotides, we con-clude that the longest correlation time reflects theinternal 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 nsin SREGGfos. The inverse of the correlation time (1 ⁄F)represents the twisting oscillation frequency (m) of thedouble helix. The oscillation frequency increases in theorder (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 whenthe temperature increases from 10 to 30 °C (notshown), indicating a lower population that depolarizesat higher temperature. Because the population of thebent form decreases at higher temperature, we mustassume that the linear form does not give a detectabledepolarization signal. Thus, fluorescence anisotropydecay mainly detects the helix twisting of the bentform offering enough thermal amplitude motions. Inaddition, b-value and thermal instability of the bentform detected using differences in Raman spectrabetween the oligonucleotides increase in the sameorder.DiscussionThe C G mutations at the )5 and )4 positions ofthe CArG box alter the binding stoichiometry in a dra-matic manner [7]. Here we show that, at 10 °C, suchmutations do not affect electric charge repartitionalong the oligonucleotides and preserve the sameB-DNA conformation. Essentially, the interactions atthe mutated positions are modified together with thearrangement of water molecules and the internaldynamics.Premelting effect on the equilibrium of the bentlinear formThe premelting transition has been studied in detail byRaman spectroscopy for alternating [poly(dA–dT)]2and homogenous poly(dA):poly(dT) sequences [30,45].The similarity to the effects of temperature on ourRaman spectra emphasizes its influence on the six cen-tral (A ⁄ T) base pairs of the CArG boxes. Detailedanalysis of Raman spectra has confirmed that theTable 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.Fans(± 0.1 ns)bb%(± 2%)m ¼1Uc106Hz NdSREGfos0.4 863.9 14 260 ± 10 1SREfos0.4 883.2 12 310 ± 10 2SREGGfos0.4 821.8 18 560 ± 20 % 4aF, correlation time. The longest correlation time characterizes theinternal motion of the DNA duplex.bb, weight of the exponentialcomponent.cm, oscillation frequency.dN, number of core-SRFmonomer bound to DNA fragment [7].J. Stepanek et al. Implication of CArG sequence in SRE flexibilityFEBS Journal 274 (2007) 2333–2348 ª 2007 The Authors Journal compilation ª 2007 FEBS 2341premelting transition conserves the basic local confor-mation features of B-DNA. (A ⁄ T)-rich sequences havebeen found to be highly polymorphic and dependstrongly on the temperature [53,54]. Indeed, the changein the array of the hydrogen bonds at thymine ofSREGGfosis probably a sign of perturbation in thehydration scheme along the minor groove of the (A ⁄ T)domain. The G–C base pair is characterized by a largedipole and both inversions change the local electriccharge repartition at )5 and )4 positions of the CArGbox, and as a consequence the interactions with watermolecules of the (A ⁄ T) domain [55,56]. Premeltingtransitions are ascribed to the disruption of watermolecules specifically bound to DNA [31,45,57]. Thepresence of a ‘low-temperature form’, referred to asB¢-type DNA, is correlated with tight binding betweenwater molecules and bases, especially in the narrowminor groove of the (A ⁄ T) domains [53,54,58].At low temperatures, between 5 and 10 °C, freeSREfosappears more bent using Raman spectroscopythan was found using electrophoresis [23,59]. Relevantto the vibrational timescale (10)14s), Raman spectros-copy allows the signals of the bent and linear conform-ers to be differentiated whatever their conversion time,whereas electrophoretic techniques average the signalsof both conformers [59]. Thus, it is more a transientbent population than a stable one that is observed insolution. From one oligonucleotide to the other two,the temperature difference Raman spectra (Figs 4–6),like the difference spectra at 10 and 25 °C (Figs 7,8),exhibit a high degree of spectral pattern conservationwith uniform low-intensity variations. The SREfosandits two mutants oscillate between a bent and a linearform keeping the same average conformations. Thus,an increase in temperature displaces the equilibrium,increasing the amplitude of motion around the regularstates within the frame of the same average geometries.These results suggest that the conversion process arisesfrom global thermal fluctuations of the oligonucleo-tides and the mutations mainly influence the probabil-ity of their occurrence [60].Bending magnitude of SREfosIn order to evaluate the bend angle induced by thedecrease in temperature from 25 to 10 °C, the Ramanspectral changes for SREfoswere compared with thoseresulting from the formation of a sharp bend in aDNA octamer duplex (HMG box) upon interactionwith the SRY(HMG) protein [47]. The CArG andHMG 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 tobe subject to a sharp bend. Moreover, the SREs usedin 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 SREfosbetween 10 and 25 °C correspond to approximatelyhalf of that caused by the SRY–HMG protein in theHMG box. Otherwise, the temperature-induced struc-tural changes in the Raman spectra during premeltingare mainly characterized, in SVD analysis, by variationin the V2contribution of the spectral component S2.Actually, the temperature profile of the V2contribu-tion is in accordance with the reduction in the bentpopulation in the oligonucleotide. Thereby, we candeduce that 10 °C corresponds closely to the tempera-ture transition between the bent form and the linearform, since their populations are roughly equivalent.The agreement between our results and those reportedby Benevides et al. [47] for the 70° sharp bend inducedin the HMG box seems very interesting. Indeed, thebend determined by Raman for the free SREfosinsolution is roughly similar to that formed in SREfosin the crystal of its complex with the core-SRF [24].This study does not provide information on the localrepartition of the angles involved in the SREfoshelixbending.Relative effect of bending strainThere are several indications of a redistribution ofthe strains exerted on the oligonucleotide by thebend: partial unstacking of some adenine, thymineand guanine bases and a more distinct presence of2¢-endo conformations of furanose rings at 10 °Cagainst a higher percentage of 3¢-endo ⁄ anti at 25 °C.Because the bend is present at low temperature, itsstabilization must be favored from the point of viewof enthalpy, but unfavored from the point of view ofentropy. A 25 °C, the higher entropy of the linearform is likely due to its higher flexibility, the highermobility of the hydration shell, or both. In thecurved 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 helicesG C base mutations at positions )4 and )5 of theCArG box induce only slight local structural differ-ences but important interactional changes betweenthe bases. The extensive empirical study of El Hassanand Calladine [56] showed that the CA step adopts awide continuous range of conformations. However,the persistence of the backbone conformation restrictsImplication of CArG sequence in SRE flexibility J. Stepanek et al.2342 FEBS Journal 274 (2007) 2333–2348 ª 2007 The Authors Journal compilation ª 2007 FEBS[...]... stabilization of the core-SRF on the consensus CArG sequence From this point of view, the various interactions connecting the bases of the CArG box play the key role in the physiological activity of DNA Experimental procedures Oligonucleotides The oligonucleotide SREfos 5¢-d(GGATGTCCATATTA GGACAT)-3¢ reproduces the sequence of the SRF recognition element of the c- fos enhancer [2,27] The mutants SREGfos 5¢-d(GGATGTgCATATTAGGACAT)-3¢... origin of the adequate conformational deformation of the CArG boxes lies in the polymorphism of the (A ⁄ T) domain Yet within the known CArG box core-SRF complexes, the CArG box remains bent whatever the mutations performed in the (A ⁄ T) sequence [23,24,26] There is a direct correlation between the degree of DNA bending and the ability of core-SRF to recognize a CArG consensus [25,26] Crystallographic... 5¢-d(GGATGTgCATATTAGGACAT)-3¢ and SREGGfos 5¢-d(GGATGTggATATTAGGACAT)-3¢ have one (C G) and two (CC GG) mutations, respectively, at the end of the CArG consensus sequence underlined above (mutations are indicated by lower case letters) HPLC-purified single strands were purchased from Invitrogen (Cergy Pontoise, France) These sequences and their complementary strands were annealed by two heating cycles... al the space devoted to the motions of this step Within the CArG box of SREfos and SREGfos, the C) 4A)3 step retains a large part of its original flexibility Conversely, the GA step adopts a restricted range of conformational space Thus, for the CArG box of SREGGfos, the rigid G) 4A)3 step inhibits (by mechanical locking) the local freedom brought about by the C) 4A)3 step in SREfos We should note that the. .. dynamical effects Previous results indicated that the dynamics of core-SRF and SREfos are crucial during complex assembly [7] The speci c recognition is in need of a particular DNA dynamic status of the bent form allowing the core-SRF to lock this transient conformer into a speci c stable bent form [23,24] Thus, the dynamics of the bent form determine when the core-SRF switch from nonspeci c to 2344 speci c. .. SREfos contribute to the speci c recognition with core-SRF The polymorphism of the (A ⁄ T) domain and the dynamics of the bent form one determinant for speci c complex formation They appear to be as important as the conservation of the DNA base sequence Therefore, the basic question is no longer what prerequisite site on the DNA determines the speci c complex formation, but rather what dynamical scenario... and linear forms depends on local SRE constraints A decrease in the strains should freeze some conversion and consequently alter the topology and the population of the bent form A greater strain in the SRE-ply enhances its rigidity, which in turn reduces the amplitude and increases the frequency of the twist If the frequency of helix twisting is too high many core-SRF gather without speci c association... conversion rates close to each other Indeed, thermal fluctuations in SREfos and SREGfos make the most of the large conformational range allowed by the C) 4A)3 step, whereas, owing to the restricted conformational space allowed by the G) 4A)3 step, SREGGfos can only take the effects of the thermal fluctuations by a higher frequency of helix Implication of CArG sequence in SRE flexibility twisting [56] The double... elastic force on the central (A ⁄ T) domain of the CArG box and on the base sequence flanking it [63] Thus, the internal dynamics of each oligonucleotide are directly correlated with its global stiffness and the behavior of the torsional oscillations reveals these changes in elasticity Only the fluorescein reporters swept by oligonucleotide molecules with sufficient stiffness experience part of the SRE internal... the C) 5C) 4 step in SREfos, the G) 5C) 4 step in SREGfos and the G) 5G) 4 step in SREGGfos belong to the class of loose steps Their conformational bistable space is not limited by mechanical locking, but by strong electrostatic interactions [56] The stiffness of the whole double helix depends on each local elasticity modulus and the strains involved between neighboring bases appear to be the main factor . C fi G base mutations in the CArG box of c- fos serum response element alter its bending flexibility Consequences for core-SRF recognition Josef. that the binding stoichiometry of core-SRF is significantlyaltered by mutations C )5 fi G (SREGfos) and C )5 C )4 fi GG (SREGGfos) of the CArG box [A
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