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© 1997 Oxford University Press 3855-3862

Sensitivity of NMR internucleotide distances to B-DNA conformation: underlying mechanics

Sensitivity of NMR internucleotide distances to B-DNA conformation: underlying mechanics Anne Lefebvre, Serge Fermandjian and Brigitte Hartmann1,*

Département de Biologie Structurale, URA 147 C.N.R.S., Institut Gustave Roussy, P.R.2, 39 rue C. Desmoulins, F-94805 Villejuif Cedex, France and 1Laboratoire de Biochimie Théorique, UPR 9080 C.N.R.S., Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, F-75005 Paris, France

Received June 27, 1997; Revised and Accepted August 12, 1997

ABSTRACT

Nuclear magnetic resonance (NMR) spectroscopy, combining correlated spectroscopy (COSY) coupling constant measurements with nuclear Overhauser effect spectroscopy (NOESY) interatomic distances, should make it possible to determine an averaged solution structure for DNA oligomers. However, even if such data could be obtained with high accuracy, it is not clear which structural parameters of DNA would be determined. Here, the relationships between measurable internucleotide distances and helical parameters are systematically studied through molecular modelling. Investigations are carried out using four representative sequences, (ACGT)n, (TCGA)n, (AGCT)n and (TGCA)n, composed of repeated tetranucleotides belonging to oligomers previously studied by NMR. Correlations between interatomic distances become evident and strong connections between distances and inter-base helical parameters are observed. Results imply that twist, roll, shift and slide values can be accurately determined from NMR data. Sequence independent mechanical coupling which link backbone and sugar conformations to helical twist are also described.

INTRODUCTION

NMR spectroscopy, associated with molecular modelling, is the only technique capable of providing a detailed molecular structure of DNA in solution. NMR allows the evaluation of certain sugar and backbone dihedral angles through coupling constant measurements and sugar puckering can generally be accurately determined (1 -5 ). Unfortunately, helicoidal parameters cannot be measured directly and must be deduced from interproton distances within or between neighbouring nucleotides. Although much effort has been put into improving the quality of interproton distance measurements (6 -8 ), it remains unclear how precisely these data determine the overall DNA structure.

Following early work onvarious aspects of this problem (9 -15 ), we attempted to carry out a systematic study using the tools offered by the JUMNA modelling program (16 ,17 ). Four repeating sequences, (ACGT)n, (TCGA)n, (AGCT)n and (TGCA)n, formed from tetranucleotides found in oligonucleotides previously studied by combined NMR and molecular modelling (4 ,18 ,19 ) were selected. These sequences contain eight out of the ten unique dinucleotide steps with examples belonging to the families YpR, RpY, YpY (where R implies purine and Y implies pyrimidine). Modelling studies have been made on helically symmetric, polymeric DNA to simplify the conformational space to be searched and to avoid end-effects. Both distances and other structural constraints were investigated through systematic scans of their values, including sugar puckering, which has already been shown to play an important role in fixing overall DNA conformation (20 ,21 ). These data were then used to search for linear correlations between different interproton distances, as well as for relationships between distances and helicoidal parameters. Obtained correlations were subsequently verified using crystallographic data.

By linking interproton distances and helicoidal parameters, this study enabled us to determine which helicoidal parameters are best defined by NMR measurements and also to identify elements of the conformational mechanics which, independently of sequence, link base and backbone movements within double helical DNA.

MATERIALS AND METHODS

All calculations were performed using the JUMNA program (16 ,17 ) which represents nucleic acid flexibility using a combination of helicoidal and internal variables. Single bond torsions and valence angles are used to model the flexibility of each nucleotide, while the nucleotides are positioned in space using helical rotations and translations with respect to a reference axis system. All bond lengths are kept fixed and the junctions between successive 3'-monophosphate nucleotides, as well as the closure of the sugar rings, are ensured using quadratic restraints on O5'-C5' and C4'-O4' distances. This representation leads to roughly ten times fewer variables than Cartesian coordinate molecular mechanics.

Helical symmetry can be imposed within JUMNA by simply making symmetry-related sets of variables equivalent to one another. This option was very useful in the present study in order to avoid the end effects associated with oligomers and to further reduce the dimension of the conformational space to be searched. Conformations were scanned using automatic adiabatic mapping options associated with distance or other structural constraints (sugar pucker, helicoidal parameters, etc.). NMR measurable interproton distances were imposed either as simple quadratic restraints with respect to a given value or as quadratic lower and upper bounds bracketing a determined range of distances.

Model and crystallographic structures were analyzed with the CURVES program (22 ,23 ), which provides a rigorous way to obtain the overall helical axis locus for irregular nucleic acids. All helical parameters were calculated with respect to this helical axis and were thus termed global parameters. All parameters obey the Cambridge convention for DNA conformation (24 ). Here, we considered intra-strand parameters, to be coherent with distances measured by NMR.

Calculations were carried out for four regular repeating polymers in the B conformation (containing phosphates in the BI conformation and sugars with `southern' puckers). The sequences studied were (ACGT)n, (TCGA)n, (AGCT)n and (TGCA)n. Systematic studies of all measurable interproton distances between nucleotides were made involving: Hx-H6/H8, Hx-H5(C) (for NpC steps, where N implies any base) and H2(A)-H1' (for ApN steps) where Hx refers to H1', H2', H2'', H3' and H6/H8 protons. Reported distances between protons are always between two sequential nucleotides and denoted by A-B, where A is a proton of the 5'-nucleotide (i) and B a proton of the 3'-nucleotide (i + 1).

Results from our modelling were compared with crystallographic data from nine B-DNA structures which were each resolved to at least 1.8 Å [below this resolution certain structural features, notably sugar puckers, become unclear (25 ,26 )]. The oligomers involved were: CCAAG*A*TTGG (27 ), CCAACITTGG (28 ), CCAACGTTGG (26 ), CGATCG[sect]ATCG (29 ), CGATTAATCG (30 ), CCAGGCCTGG (31 ), CCAG#CGCTGG (32 ), CCAGGCmeCTGG (33 ) and C[sect]GATATATCG (34 ). Junctions perturbed by mismatches (G*A*), unusual bases (G#: oxoguanine) or unusual backbone conformations ([sect]: [alpha]/[beta]/[gamma] crankshafts) were not considered, but all others were taken into account, including sugars having phase angles between 80 and 200o or positive [epsilon]-[zeta] angle differences, since such conformations are known to exist in solution. Since decamers within crystal stack end-to-end to form continuous double helices, terminal base pairs were also considered. Given these restrictions, the nine oligomers contain a total of 116 non-redundant single strand dinucleotide steps.

RESULTS

Energy mapping and parameter variability

For each of the four repeating sequences studied, the conformational space was explored by scanning the sugar phase angles from 130 to 200o. Because of inversion symmetry each of the tetranucleotide repeats studied has four unique sugars, but, because the puckers of successive sugars are coupled to one another, the search can actually be simplified to mapping the phase angle differences between two successive pairs of nucleotides belonging to one strand of the duplex. We have previously demonstrated that this approach enables us to locate all the stable B-family conformations of such sequences (4 ,19 ,21 ,35 ). The energy map for (ACGT)n presents three distinct stable conformations, whereas (AGCT)n and (TGCA)n have two minima and (TCGA)n only one (see Table 1 ).

Starting from each of these minima, we carried out mapping of the interproton distances relevant to NMR spectroscopy for the internucleotide steps of each sequence. Distances >4 Å were varied over a 3 Å range and distances <4 Å over a 2 Å range. An example of the results obtained is given in Figure 1 B for the H6-H8 distance of the CpG step and the H3'-H8 distance of the TpC step within the (TCGA)n sequence. The variation of interproton distances over quite large ranges gave rise to only small changes of energy (of the order of a few kcal/mol), as was the case for individual sugar phase angle variations (see Fig. 1 A). However, it was noted that longer internucleotide distances, such as H6/H8-H6/H8, were generally more easily variable than shorter distances, such as H2''-H6/H8 (Table 2 ). The variability of longer distances was also more susceptible to sequence effects. This observation is illustrated by H6/H8-H6/H8 distances, for which RpY steps were the most rigid (0.8-1.0 Å variation for an energy change of 1 kcal/mol), followed by RpR and YpY steps (1.0-1.2 Å variation) and finally YpR steps (1.2-1.6 Å variation). It is interesting to remark that within the latter family, CpA and CpG were the most deformable steps, illustrating once again their conformational malleability (4 ,19 ,28 -30 ,36 -38 ). Shorter internucleotide distances showed much less sequence effects, although for H3'-H6/H8 it was possible to distinguish YpR and RpR steps (0.7-1.0 Å variation) from more rigid YpY and RpY steps (0.5-0.7 Å variation).

Scans of purine phase angles were also carried out. The conformation of these sugars have the largest impact on overall DNA conformation and, in the case of the sequences studied, allow all the minimal energy conformations to be reached along a single phase angle pathway (see examples in Fig. 1 A). The interproton distance and phase angle scans carried out generated considerable variations in most of the helicoidal parameters (Table 3 ). However, direct scans of helicoidal parameters were also performed for the (ACGT)n sequence, in order to verify the relationships determined from the distance and phase angle mapping, as well as to generate structures with more extreme values of the helicoidal parameters. This was necessary, for example, for tilt angles, which otherwise varied only weakly (see Table 3 ), as also observed for crystal structures (39 ). The energy cost of helicoidal deformations depended strongly on the type of parameter involved, X displacement, inclination and rise being much softer variables than twist, roll, shift or tip parameters (see examples in Fig. 1 C and D).

Table 1 Energies (kcal/mol) and sugar phase angles (o) per unit cell for the stable sub-states conformations for the tetranucleotide repeat polymers studied
SequenceEnergySequenceEnergy
ACGT P(A)P(C)P(G)P(T)TCGA: P(T)P(C)P(G)P(A)
1-199.61521661621651-198.5156167155170
2-199.5153167178161      
3-198.4182152181162      
TGCA: P(T)P(G)P(C)P(A)AGCT: P(A)P(G)P(C)P(T)
1-202.71551491651801-203.5154153167164
2-202.21521791571842-202.7180148168164

Table 2 . dav (Å), average values of Hx-H6/H8 internucleotide distances; [Delta](dav) (Å), average variations for an energy cost of 1 kcal/mol; [Delta]([Delta]d)seq (Å), sequence variability of these variations
DistancesH1'-H6/H8H2'-H6/H8H2''-H6/H8H3'-H6/H8H6/H8-H6/H8
dav 3.43.52.34.85.0
[Delta](dav)0.81.00.60.81.2
[Delta]([Delta]d)seq0.40.40.0.40.8
Sequential distances are noted by A-B, where A is a proton of the 5'-nucleotide (i) and B a proton of the 3'-nucleotide (i + 1).

Table 3 . Helical parameter ranges of the structures generated with purine phase angle and internucleotide distances scans
Inter baseXdisp Ydisp Inc. Tip   
min./max.-2.5/0.-0.4/0.4-15/5.0-8./8.  
Inter base pairShift Slide TiltRollTwistRise
min./max. -1./1.-1./1.-4./4.-12./15.29./44.3./4.
SugarPhaseAmp.    
min./max. 140./195.34./46.    
Xdisp, Ydisp, shift, slide (Å); Inclination, tip, tilt, roll, twist, rise, phase and amplitude (o).


Figure 1.Deformation energy d(E) (kcal/mol) associated with variations of: (A) guanine phase angle of (TCGA)n: ______ and of (TGCA)n: - - - - ; (B) H6/H8-H6/H8 distance at the CpG step: ______ and H3'-H6/H8 distance at the TpC step: - - - - for the (TCGA)n sequence; (C) inclination of the CpG step of (ACGT)n; (D) shift of the CpG step of (ACGT)n. Reported sequential distances are noted by A-B, where A is a proton of the 5'-nucleotide (i) and B a proton of the 3'-nucleotide (i + 1).

About 3000 structures were generated in this way. It should be noted that none of the conformations generated left the B-DNA family by adopting sugar pucker or backbone conformations other than those known to predominate in solution.

Relationships involving the internucleotide distances H2'-H6/H8 and H6/H8-H6/H8

We now consider the relationship between the distances H2'-H6/H8 and H6/H8-H6/H8. These two distances were found to vary in a coupled manner, in contrast to the H1'-H6/H8 and H3'-H6/H8 distances which varied independently. The coupling is illustrated in Figure 2 A, which additionally shows that our model conformations and the crystallographic conformations lead to the same relationship, although the crystal structures cover a wider variation of interproton distances. It is also remarked that this relationship was found to hold even for non-canonical DNA conformations involving O4'-endo sugars or BII backbone conformations.


Figure 2.(A) Correlation between the H2'-H6/H8 and H6/H8-H6/H8 internucleotide distances; (B) H6/H8-H6/H8 distance calculated from the equation De = 0.06 * Twist - 0.04 * Roll - 0.4 * Shift + 3.0 versus the measured distance, D; (C) H2'-H6/H8 distance calculated with the equation De = -0.02 * Roll - 0.15 * Shift +0.07 * Amplitude5' as a function of the measured distance, D. (-, model structures generated with JUMNA; -, crystal structures).

The coupling demonstrated between the H2'-H6/H8 and H6/H8-H6/H8 distances can be useful in verifying the coherence of NOESY data since the linear correlation between them holds to a precision of ~0.4 Å for the B-DNA family. Points beyond this range must be suspected of errors in measurement (assignment error, low signal to noise ratio, peak overlap, or other errors related to NOE volume measurement), and imply very distorted and unusual conformations.

Relationships between helicoidal parameters and Hx-H6/H8 distances

In addition to the observation of a direct correlation between the H2'-H6/H8 and H6/H8-H6/H8 distances, these distances can be derived from the corresponding helicoidal parameters, as shown by the equations below:
DH6/H8-H6/H8 = 0.06 Twist -0.04 Roll -0.4 Shift +3.01
DH2'-H6/H8 = 0.05 Twist -0.01 Roll -0.3 Shift +2.0?2

Note that, the H6/H8-H6/H8 distance (1), which does not involve sugar protons, was logically found to depend only on parameters describing the relative position of the two bases of interest, namely the twist, shift and roll. We tested the validity of these equations by calculating DH6/H8-H6/H8 and DH2'-H6/H8 from the helicoidal parameters, according to equations 1 and 2, and comparing these values with the measured distances (Fig. 2 B and C). This was performed for all structures calculated with the JUMNA program, as well as for the crystal structures. Note that from our equation the H6/H8-H6/H8 distances show a particularly good fit with crystal structures distances. In relation, Hahn and Heinemann (25 ) have shown that within a crystal structure refined to 1.7 Å, the base positions are well defined, whereas sugar phosphate backbone angles are more influenced by refinement program.

These equations allow estimation of twist and roll parameters on the basis of interproton distances. For moderate values of base pair shift, as shown in Figure 3 A and C, or, if twist is evaluated using phosphorus chemical shift data (4 ), they can also yield to accurate roll and shift values.


Figure 3. Contour lines of Hx-H6/H8 internucleotide distances calculated as a function of helical parameters by equations in Table 4. (A) H6/H8-H6/H8 for Shift = +0.5 Å (left) and -0.5 Å (right); (B) H1'-H6/H8; (C) H2'-H6/H8 for Roll = 0; (D) H3'-H6/H8 for Amplitude5' = 39o.

It is remarked that it is also possible to express the H2'-H6/H8 distance as a function of roll, shift and sugar amplitude (Table 4 ),DH2'-H6/H8 = -0.02 Roll -0.15 Shift +0.07 Amplitude5'3

The appearance of the sugar amplitude in the above equation recalls that for a sugar in the C2'-endo conformation, the position of the H2' proton is very sensitive to amplitude variations. This again illustrates the close connections between sugar pucker and base movement within the DNA double helix (4 ,20 ,21 ,40 ,41 ). It specifically reflects the correlation previously found between the helical twist of a dinucleotide step and the amplitude of the 5'-sugar as illustrated for the present set of sequences in Figure 4 .


Figure 4.Correlation between 5' sugar amplitude and twist angle.

In the same way, we note that slide is also well determined, due to a strong correlation with twist parameter. A similar coupling has been drawn in previous studies (13 ,20 ,29 ).

Similar relationships may be obtained for the internucleotide distances H1'-H6/H8 and H3'-H6/H8 and all of the linear equations established from the present study are summarised in Table 4 .

Table 4 . Coefficients of the best linear equations determining Hx-H6/H8 internucleotide distances as a function of helical parameters and the accuracy (Å) observed in back-calculations
 TwistRollShiftPhaseAmp.ConstantDispersion
H6/H8-H6/H8 0.06-0.04-0.4  3.0"0.3
H2'-H6/H8 0.05-0.01-0.3  2.0"0.3
  -0.02-0.15  0.071.0"0.2
H1'-H6/H8   -0.01-0.067.5"0.2
H3'-H6/H8-0.04  0.1  0.025.5"0.2

Concerning H1'-H6/H8 distances, we observed that, in our structures, the H1'-H6/H8 vector is, on average, almost parallel to the helix axis, this position corresponding to a minimal value of the H1'-H6/H8 distance (Fig. 5 ). When helicoidal parameters were varied, the distance increased in a roughly quadratic manner. As a consequence, linear coefficients in the distance/helicoidal parameters relationships being close to zero and helicoidal parameters such as twist and shift have only a minor effect of this distance. It was found possible to obtain a regression equation based solely on the 5' sugar conformation, which gave H1'-H6/H8 values to a precision of +-0.2 Å for the model conformations (Table 4 ) and could also be applied to the crystal data. This distance is consequently a good indicator of the 5' sugar conformation of a dinucleotide step (Fig. 3 B) and it may again be used to check the coherency of the NMR data concerning DNA oligomers.


Figure 5.Trajectories of the H1' (- - - - ), H2'' (_ _ _ _ ) and H2' (- . - . - . -) protons, projected in the plan orthogonal to the C68-H6/H8 chemical bond. Note that H1' trajectory was contained in this plane, and that H2' and H2'' did not move from more than 20-30o from this plane. The plain straight line represents the plane of the base, and squares indicate the atom average positions. Note the contrast between H2' position and H2'' or H1' one. These two latter protons are moving around a position corresponding to a minimal H1'-H6/H8 or H2''-H6/H8 distance (represented by the vertical line on the scheme), whereas the H2' proton never adopts such a position with respect to the H6/H8 proton.

The H3'-H6/H8 distance undergoes only small variations (no more than 0.6 Å within our structures). Yet, it demonstrated the dependence on twist, shift and 5' sugar amplitude. Since twist variations dominate the correlation, this distance can be used as a twist indicator. For example, a H3'-H6/H8 NOE intensity clearly stronger than the average may well be associated with a high twist value. This distance is the only one belonging to the Hx-H6/H8 family which decreases with increasing twist (Fig. 3 D). Thus, a large twist which can lead to the disappearance of H6/H8- H6/H8 and H2'-H6/H8 NOE cross-peaks may simultaneously create an intense H3'-H6/H8 NOE cross-peak.


Figure 6.Distribution of the number of H2''-H6/H8 internucleotide distances (N) versus H2''-H6/H8 values in: (A) all steps of crystal structures (116 distances); (B) without BII steps [([epsilon]-[zeta]) > 20o, 74 distances].

Lastly, we considered the H2''-H6/H8 distance which varies by only 0.5 Å within the conformations we have generated (see also, ref. 13 ). Although it varies proportionally like the other distances (~25%), its shortness (centered on 2.3 Å) leads to small absolute variations. Moreover, the H2'' proton occupies a position similar to that of H1' proton (see Fig. 5 ), rendering the H2''-H6/H8 distance rather insensitive to helicoidal parameter variations. Crystallographic data (Fig. 6 ) support this finding for canonical backbone BI conformations (Fig. 6 B), although the presence of BII phosphate conformations leads to considerably increased values. This effect also underlies the sequence dependent variations of this distance discussed by Fedoroff et al. (42 ). These latter authors interpreted variations of up to 2.7 Å for YpR and RpR steps in terms of `profile sums'. Large H2''-H6/H8 distances were found associated with extreme values of helicoidal parameters. BII backbone conformations, which occur preferentially at YpR and RpR steps and induce large changes in inter base pair helicoidal parameters (notably when they occur in both strands of the duplex) are at the origin of such effects (19 ,40 )

Table 5 . Coefficients of the best linear equations determining Hx-H5 internucleotide distances as a function of helical parameters
 XdispInc.TipTwistRollShiftPhaseAmp.Constant
H6/H8-H5    0.04-0.04-0.2   2.6
H1'-H50.35 -0.05-0.1  -0.01 10
  -0.03-0.07-0.1  0.55-0.01  9.3
H2'-H5 -0.02-0.025-0.075   0.06 3.8
H2''-H50.4 -0.025-0.15  -0.01 10.7
  -0.04-0.06-0.15  0.6-0.01 10.3
H3'-H5 -0.02-0.03-0.1  0.4 0.06 7.25
The accuracy observed in back-calculations is between "0.2 and "0.25 Å for each distance.

To summarise these results, twist, slide, shift and roll are largely involved in Hx-H6/H8 distances values. Rise does not appear in our relationships, but it generally varies relatively weakly and large changes necessarily seem to imply, and to be coupled to, large changes on other parameters. Tilt is not easily determined, but also does not vary very strongly, as shown in crystallographic structures. We can note that Hx-H6/H8 distances were not very sensitive to the base orientation around the glycosidic bond (such as inclination or tip) as the H6 and H8 protons are located very near the C1' pivot. We can thus predict that distances related to protons more distant from the pivot, such as H5-cytosine or H2-adenine, will be better indicators of such rotations.

Relationships involving internucleotide distances with H5(C)

The cytosine H5 proton is submitted roughly to the same movements during DNA conformational change as its close neighbour H6 proton. Consequently, Hx-H5 distances vary as the corresponding Hx-H6/H8 distances discussed above. This notably concerns internucleotide distances where Hx is H5 or H6/H8 (see Fig. 7 A), H1' and, to a lesser extent, H3' protons. Thereby, for steps with a cytosine in the 3' position, coherence between the Hx-H6 and the associated Hx-H5 distances should be verified.


Figure 7. (A) Correlation between H6/H8-H6/H8 and H6/H8-H5 internucleotide distances; (B) correlation between H2''-H5 and H1'-H5 internucleotide distances. -, Structures generated with JUMNA; -, crystal structures (22 distances).

A number of correlations were found amongst Hx-H5 distances, and, for example, H1'-H5 and H2''-H5 distances are strongly coupled (Fig. 7 B). The H3'-H5 distance was found to be correlated to both the H2'-H5 and H2''-H5 distances, although it is too long (>5 Å) to be of interest for NMR spectroscopy. Table 5 summarises the linear equations relating H5 distances to helicoidal and backbone parameters. Distance/helicoidal parameter correlations for H6/H8-H5 and H2''-H5 are illustrated in Figure 8 A and B. Unlike the H1'-H6/H8 and H2''-H6/H8 vectors, H1'-H5 and H2''-H5 vectors are not parallel to the helix axis, rendering them much more sensitive to helicoidal parameters variations. Note that for the related H2''-H5 and H1'-H5 distances, two equations were determined, the first including the X displacement, and the second, shift and inclination. This reflects the correlation between the X displacement and the inclination, already suggested by the work of Boutonnet et al. (43 ).


Figure 8. Contour lines of Hx-H5 internucleotide distances calculated as a function of helical parameters by equations in Table 5. (A) H6/H8-H5 for Shift = +0.5 Å (left) and -0.5 Å (right); (B) H2''-H5 for Tip = 0o and phase angle = 160o.

As expected, more parameters corresponding to rotations are involved in Hx-H5 distances than in Hx-H68 distances. Nevertheless, twist and shift parameters are still dominant, explaining the correlations observed between H1'-H5 and H1'-H6/H8 distances or between H3'-H5 and H3'-H6/H8 distances. Since these equations involve more than three parameters they cannot be used for directly determining individual helicoidal parameters. The corresponding interproton distances are nevertheless important in fixing DNA conformation, as they are the only ones to be linked to base inclination and tip.

Table 6 . Coefficients of the best linear equations determining HC2-H1' internucleotide distances as a function of helical parameters
 XdispYdispIncTwistPhaseConstant
HC2-H1'0.15-0.30.03-0.040.022.85
The accuracy observed in back calculations is "0.1 Å.

Note that all the correlations between helicoidal parameters and distances discussed here concern single strand parameters. It should not be forgotten, that within DNA oligomers with irregular conformations, there may be not negligible differences between the two strands of a given dinucleotide step. Therefore, irregular single strand base-axis parameter measurements determine double strand parameters values such as buckle or propeller-twist.

Relationships involving internucleotide H2(A)-H1' distances

This distance behaved in a completely different manner with respect to other distances discussed. This is due to the fact that the adenine H2 proton lies roughly in the centre of the minor groove of the double helix. It is also found for longer distances than any other proton considered from the glycosidic bond linking the base to the backbone. Thus, the H2-H1' distance is under the dependence of a large number of parameters (Table 6 ), and cannot be used to evaluate any individual helicoidal parameter. It is, however, certainly of interest for accurately positioning adenine bases within the duplex conformation.

CONCLUSIONS

How precisely a B-DNA structure can be defined from a joint NMR spectroscopy and molecular modelling still remains a difficult question to answer. When interproton distances are the only constraints imposed, many differences are observed amongst the calculated structures, these clearly depending on the starting conformations and, obviously, the quality of methods applied to determine the distance constraints from NOE intensities. Generally, a good consensus can be obtained for a set of parameters limited to the helical twist and, eventually, the roll angle (2 ,9 -11 ). Ulyanov et al. (2 ) has clearly posed the question of how uniquely NMR data determine DNA conformation. By generating structures in which one helicoidal parameter was systematically changed, these authors have observed that some distances vary much more than others (notably, internucleotide H6/H8-H6/H8 distances). However, due to mechanical compensations, the global R factor with respect to a given set of experimental interproton distances remained almost constant. This factor is consequently a poor guide for discriminating between different conformations. Convergence between conformations can be improved by applying time average restraints (44 ,45 ) or by adding restraints on sugar puckers and backbone angles (46 ,47 ). The latter strategy has been used effectively in conjunction with distance constraint refinement against NOE data, in previous studies with the JUMNA program, allowing us to obtain consensus structures for a number of oligonucleotides (4 ,18 ,35 ). These studies have further shown that considering local R-factors, corresponding either to one given step or to one distance type, leads to a better estimation of the agreement with NOE data than a single, global R-factor. Yet, if we assume in an ideal case that all accessible distances could be determined accurately from NMR data, how many helicoidal parameters are really fixed by these distances?

The aim of the present study was to systematically investigate the relationships between all measurable internucleotide distances and helicoidal parameters in order to determine which sort of parameters were dominant and, consequently the role of distances in fixing overall helicoidal conformation. The results obtained for four repeating tetranucleotide sequences by restrained energy mapping, show that amongst the three rotation (twist, roll and tilt) and the three translation (shift, slide and rise) parameters which define the structure of a dinucleotide step, twist is the most easily accessible, since it is virtually connected to all the internucleotide distances. The roll and shift parameters appear to be relatively well constrained for similar reasons. The slide is also well determined, not because it is directly related to any given distances, but rather for its strong correlation with helical twist. Rise and tilt are not easily determined, but also do not vary very strongly.

Distances involving base protons located far from the glycosidic bond such as H5(C) or H2(A) are the most sensitive to the rotational base-axis parameters. Thus, parameters which fix base position with respect to the helical axis are only clearly given by distances involving adenine or cytosine protons and consequently, are sequence dependent.

This study also reveals the general mechanics of the B-DNA double helix. A number of clear correlations exist and reflect helical parameters coupling. The correlations between interatomic distances and the fact that certain distances can be expressed by two different sets of helical parameters lead to mechanical coupling between helicoidal parameters. The particular coupling between twist and sugar amplitude described here stresses the link which exists between base movements and backbone conformation via the puckering amplitude of the 5" sugar of each dinucleotide step and the related correlation between the twist and the backbone [epsilon]-[zeta] angle difference. Such mechanical coupling appears to be largely sequence independent and remains valid even for non-canonical sugar puckers and backbone conformations.

ACKNOWLEDGEMENTS

A. Lefebvre is the fellowship of the Institut de Formation Supérieure Biomédicale. B. Hartmann wishes to thank the Association for International Cancer Research (St Andrews, UK) for their support. This work was also partially supported by the Association pour la Recherche contre le Cancer and the Ligue Nationale Française contre le Cancer. The authors also wish to thank Muriel Delepierre and Richard Lavery for helpful discussions.

REFERENCES

1 Schmitz, U., Sethson, I., Egan, W.M. and James, T.L. (1992) J. Mol. Biol. 227, 510-531. MEDLINE Abstract

2 Ulyanov, N.B., Schmitz, U., Kumar A. and James, T.L. (1995) Biophysical J. 68, 13-24.

3 Gaudin, F., Paquet F., Chanteloup, L., Beau, J.M., Nguyen, T.T. and Lancelot G. (1995). J. Biomol. NMR 5, 49-58. MEDLINE Abstract

4 Lefebvre, A., Mauffret, O., Hartmann, B., Lescot, E. and Fermandjian, S. (1995) Biochemistry 34, 12019-12028. MEDLINE Abstract

5 Conte, M.R., Bauer, C.J. and Lane, A.N. (1996) J. Biomol. NMR 7, 190-206. MEDLINE Abstract

6 Wijmenja, S.S., Mooren, M.M.W. and Hilbers, C.W. (1993) In Roberts, G.C.K. (ed.) NMR of Macromolecules. IRL Press, Oxford, pp. 217-283.

7 James, T.L. (1994) Curr. Opin. Struct. Biol. 4, 275-284.

8 Liu, H., Spielmann, H.P., Ulyanov, N.B., Wemmer, D.E. and James, T.L. (1995) J. Biomol. NMR 6,390-402. MEDLINE Abstract

9 Metzler, W.J., Wang, C., Kitchen, D.B., Levy, R.M. and Pardi, A. (1990) J. Mol. Biol. 214, 711-736. MEDLINE Abstract

10 Lane, A.N. (1990) Biochim. Biophys. Acta 1049, 205-212. MEDLINE Abstract

11 Kaluarachchi, K., Meadows, R.P. and Gorenstein, D.G. (1991) Biochemistry 30, 8785-8797. MEDLINE Abstract

12 Schmitz, U., Pearlman, D.A. and James, T.L. (1991) J. Mol. Biol. 221, 271-292. MEDLINE Abstract

13 Ulyanov, N.B., Gorin, A.A., Zhurkin, V.B., Chen, B.-C., Sarma, M.H. and Sarma, R.H. (1992) Biochemistry 31, 3918-3930. MEDLINE Abstract

14 Leijon, M., Zdunek, J., Fritzsche, H., Sklenar, H. and Graslund, A. (1995) Eur. J. Biochem. 234, 832-842. MEDLINE Abstract

15 Schmitz, U. and James T.L. (1995) Methods Enzymol. 261, 3-45. MEDLINE Abstract

16 Lavery, R., Zakrzewska, K. and Sklenar H. (1995) Comp. Phys. Comm. 91, 135-158.

17 Lavery, R. (1988) In Olson, W.K., Sarma, R.H., Sarma, M.H. and Sundaralingam, M. (eds) Structure and expression. Vol.3, DNA bending and curvature. New York, Adenine Press. pp. 191-211.

18 Mauffret, O., Hartmann, B., Convert, O., Lavery, R. and Fermandjian, S. (1992) J. Mol. Biol. 227, 852-862. MEDLINE Abstract

19 Lefebvre, A., Mauffret, O., Lescot, E., Hartmann, B. and Fermandjian, S. (1996) Biochemistry 35, 12560-12569. MEDLINE Abstract

20 Poncin, M., Hartmann, B. and Lavery R. (1992) J. Mol. Biol. 226, 775-794. MEDLINE Abstract

21 Lavery, R. and Hartmann, B. (1994) Biophys. Chem. 50, 33-45. MEDLINE Abstract

22 Lavery, R. and Sklenar, H. (1988) J. Biomol. Struct. Dynam. 6, 655.

23 Lavery, R. and Sklenar, H. (1989) J. Biomol. Struct. Dynam. 6, 655-661.

24 Dickerson, R.E., Bansal, M., Calladine, C.R., Diekmann, S., Hunter, W.N., Kennard, O., Lavery, R., Nelson, H.C.M., Olson, W.K., Saenger, W., et al. (1989) J. Mol. Biol. 205, 787-791.

25 Hahn, M. and Heinemann, U. (1993) Acta Cryst. D49, 468-477.

26 Privé, G.G., Yanagi, K. and Dickerson, R.E. (1991) J. Mol. Biol. 217, 177-199. MEDLINE Abstract

27 Privé, G.G., Heinemann, U., Chandrasegaran, S., Kan, L.S., Kopta, M.L. and Dickerson, R.E. (1987) Science 231, 498-504.

28 Lipanov, A., Kopka, M.L., Kaczor-Grzeskowiak, M., Quintana, J. and Dickerson, R.E. (1993) Biochemistry 32, 1373-1389. MEDLINE Abstract

29 Grzeskowiak, K., Yanagi, K., Privé, G.G. and Dickerson, R.E. (1991) J. Biol. Chem. 266, 8861-8883. MEDLINE Abstract

30 Quintana, J.R., Grzeskowiak, K., Yanagi, K. and Dickerson, R.E. (1992) J. Mol. Biol. 225, 379-395. MEDLINE Abstract

31 Heinemann, U. and Alings C. (1989) J. Mol. Biol. 210, 369-381. MEDLINE Abstract

32 Lipscomb, L.A., Peek, M.E., Morningstar, M.L., Verghis, S.M., Miller, E.M., Rich, A., Essigmann, J.M.and Williams, L.D. (1995) Proc. Natl. Acad. Sci. USA 3, 719-723.

33 Heinemann, U. and Hahn, M. (1992) J. Biol. Chem. 267, 7332-7341. MEDLINE Abstract

34 Yuan, H., Quintana, J. and Dickerson, R.E. (1992) Biochemistry 31, 8009-8021. MEDLINE Abstract

35 Sodano, P., Hartmann, B., Rose, T., Wain-Hobson, S. and Delepierre, M. (1995) Biochemistry 34, 6900-6910. MEDLINE Abstract

36 Timsit, Y., Vilbois, E. and Moras D. (1991) Nature 354, 167-170. MEDLINE Abstract

37 El Antri, S., Bittoun, P., Mauffret, O., Monnot, M., Convert, O., Lescot, E. and Fermandjian, S. (1993) Biochemistry 32, 7079-7083. MEDLINE Abstract

38 Timsit Y. and Moras D. (1995) J. Mol. Biol. 251, 629-647. MEDLINE Abstract

39 Gorin, A.A., Zhurkin V.B. and Olson W.K. (1995) J. Mol. Biol. 247, 34-48. MEDLINE Abstract

40 Hartmann, B., Piazzola, D. and Lavery, R. (1993) Nucleic Acids Res. 21, 561-568. MEDLINE Abstract

41 Sarai, A., Jernigan, R.L. and Mazur, J. (1996) Biophys. J. 71, 1507-1518. MEDLINE Abstract

42 Fedoroff, O.Y., Reid, B.R. and Chuprina, V.P. (1994) J. Mol. Biol. 235, 325-330. MEDLINE Abstract

43 Boutonnet, N., Hui, X. and Zakrzewska, K. (1993) Biopolymers 33, 479-490. MEDLINE Abstract

44 Pearlman, D.A. and Kollman, P.A. (1991) J. Mol. Biol. 220, 457-479. MEDLINE Abstract

45 Torda, A.E., Scheck, R.M. and van Gunsteren, W.F. (1989) Chem. Phys. Lett. 157, 289-294.

46 Kim, S.-G. and Reid, B.R. (1992) Biochemistry 31, 12103-12116. MEDLINE Abstract

47 Kim, S.-G., Lin, L.J. and Reid, B.R. (1992) Biochemistry 31, 3564-3574. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +33 1 43 25 26 09; Fax: +33 1 43 29 56 45; Email: brigitte@ibc.fr
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