Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 10, Issue Number 3 (1992), ®Adenine Press (1992).
A High Angle Neutron Fibre Diffraction Study of the Hydration of the A Conformation of the DNA Double Helix P. Langan\ V.T. Forsyth\ A. Mahendrasingam\ W J. Pigram\ S.A. Mason 2 and W. Fuller1 1
Department of Physics Keele University Staffordshire, ST5 5BG
2
Institut Laue-Langevin B.P. 156 38042 Grenoble Cedex 9, France Abstract A high angle neutron fibre diffraction study of the distribution of water around the A-form of DNA has been performed using the diffractometer Dl9 at the Institut Laue-Langevin, Grenoble. These experiments have exploited the ability to replace H 20 surrounding the DNA by D 20 so that isotopic difference Fourier maps can be computed in which peaks are identified with the distribution of water in the unit cell. All peaks of significant height have been accounted for by four families of water molecules whose positions and occupancies have been determined using least squares refinement. The coordinates of the water peaks making up each family do not deviate significantly from a regular helical arrangement with the same parameters as the DNA Two of these families are of particular intererst. The first consists of water molecules in the major groove linking successive charged phosphate oxygens along the polynucleotide chains. The second is associated with bases in the major groove and forms a central core of density along the helix axis. These two families provide a layer of hydration lining the interior wall of the major groove leaving a central channel to accommodate cations. The relationship between these observations and conformational stability is discussed.
Introduction Deoxyribonucleic acid (DNA) is a double-helical polymer which has been shown by x-ray fibre diffraction to assume a number of distinct conformations depending on hydration, ionic environment, base sequence and nearest neighbour interactions ( 1, 2). These studies have provided detailed molecular models for the A, B and C forms, the three conformations commonly available to naturally occurring DNA (3-5). The synthesis of polynucleotides with repeating base pair sequences has led to the observation of novel forms such as D for poly[ d(A-T)]. poly[d(A-T)] (6) and S (or Z) for poly[d(G-C)).poly[d(G-C)) (7, 8) as well as variants of the B form such as B"
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for poly[d(G-C)].poly[d(G-C)] (9). By exploiting the availability of the high brightness Synchrotron Radiation Source at the SERC Daresbury Laboratory we have performed time-resolved x-ray fibre diffraction studies of structural transitions in DNA(I0-12). Most synthetic and naturally occurring DNAs can assume A-type conformations in fibres where the counter ion is Na +, K+, Rb +orCs+ but not Li+, at relative humidities in the range of 66%-86%. The A-form is an 11-fo1d right-handed double helix with a pitch of 28.03A, crystallising in the monoclinic space group C2 with a=21.7A, b=39.9A, c=28.03Aand ~=96.82° (13). In early fibre diffraction experiments the Aform was observed to undergo a reversible structural transition to the B-form by increasing the relative humidity from 75% to 92% (I). Although the minor groove in B-ONA is significantly narrower than the major groove both grooves have similar depths. The displacement of the base pairs in A-DNA by about SA from their position in B-ONA away from the helix axis towards the minor groove creates a shallow minor groove and a deep major groove which in projection down the helix appears as a 6A diameter hole. Information on the hydration of DNA has come from x-ray diffraction studies of short fragments of single crystal oligonucleotide duplexes. Whereas fibre diffraction studies emphasise regularity in the double helix and factors associated with cooperative behaviour, single crystal studies highlight local variation as a function of base pair sequence. Although these techniques are complementary, care must be taken in relating results from oligonucleotides to DNA because of end effects stemming from the limited length ofthe oligonucleotide and the presence of crystallising agents. In the first A-type tetramer fragment to be studied a network of water bridging the major groove was found which Conner eta/ (14) suggested might be important in stabilising the A conformation. Similar studies on oligonucleotide duplexes in the B and Z forms have revealed an apparent correlation between the degree of extension of the DNA strands and hence the separation of adjacent phosphates and the number of water molecules associated with each phosphate group. This observation led Saenger eta/ (15) to propose that DNA conformation is determined by economy in the hydration of phosphate groups. The validity of this extrapolation of observations on oligonucleotides to polymeric DNA has been discussed (16, 17). The substantially greater scattering length of 0 20 for neutrons as compared to xrays and the possibilities for H 20/D20 isotopic substitution offer important advantages for locating water around DNA by using neutron rather than x-ray diffraction. In earlier neutron fibre diffraction studies we have exploited the difference in the scattering lengths of hydrogen and deuterium to locate water around the D-form of DNA (18, 19). Hydration patterns observed in these studies are stereochemically consistent with information from x-ray fibre diffraction studies on the location of ions within the same DNA structure (20). We describe here how these Fourier difference synthesis techniques based on the isotopic substitution of Hp by Dp in fibres of DNA have allowed us to image scattering density associated with localised water around the A-DNA double helix.
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Materials and Methods
Commercially purchased E.coli DNA was purified using previously described techniques (21) and the ionic content controlled so that fibres could be drawn which at 75% relative humidity reproducibly assumed the A conformation. Diffraction data were recorded using the single crystal diffractometer D 19 at the Institut LaueLangevin, Grenoble, with a wavelength of2.42A (19). The D19 detector is banana shaped with an aperture of 64 o X4 o, mounted with its long axis vertical so that it could be rotated about a vertical axis through the specimen. In order to achieve the desired sample size for neutron fibre diffraction studies a cuboid sample 1X5X5mm was constructed from -100 fibres each -2001J diameter and -5mm long mounted on a mica sheet. The sample was housed in a cylindrical can lOcm in diameter and 18cm in length with Melinex windows of0.036mm thickness. The relative humidity was controlled by passing helium, humidified to 75% by bubbling through an appropriate saturated salt solution, through this can. Exchange of H 20 by D 20 in the sample was achieved by replacing the salt solution by one where the solvent was D 20 and was typically complete over a period of6-8 hours. H 20/D20 exchange was monitored by observing changes in the coherent and incoherent scattering. Scattering from a standard vanadium rod was measured at the start and end of each run to calibrate the detector. Data to a resolution of3A were collected as 21 partially overlapping detector frames using an angular increment of 2.5°. Software was developed to construct a composite image of the diffraction data by merging these frames after stripping off overlapping regions to minimise edge effects. A collection time of 4 hours was required in each detector position for adequate intensity statistics. The data at each position were collected in two separate scans across the diffraction image to confirm experimental reproducibility. Figure 1 shows the images remapped into flat film geometry so thatthe distribution ofintensityis readily compared with that of typical x-ray patterns. The substantial differences in the intensity of corresponding diffraction peaks when DNA is surrounded by D 20 and H 20 are illustrated by the difference image constructed by subtracting one image from the other.
Figure 1: Neutron fibre diffraction data: (a) A-DNA surrounded by HP and (c) the difference between (a) and (b).
Dp, (b) A-DNA surrounded by
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9
8
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6
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s
+----------------------------~Ld~------------------------
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3
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0. 02
0. 04
0. 06
0. 08
O. 10
0. 12
D. 1~
O. 16
0. 18
D. 2D
D. 22
Reciprocal spacing (A- 1 ) Figure 2: Comparison of the observed structure factor amplitudes corresponding to DNA surrounded by Hp (full bars) and DNA surrounded by DP (open bars). Overlaid bars correspond to accidently overlapping reflections.
Scattering from the Melinex windows was allowed for by recording diffraction with the sample removed. Once the background baseline had been subtracted from the observed diffraction patterns, Bragg intensities were determined by fitting Gaussian profiles using purpose-designed software. These intensities, I0(h). were corrected for the Lorentz effect and for attenuation using a transmission factor with a mean effective linear absorption coefficient, f.l, dominated by the effective absorption cross-section ofhydrogen, crh. In order to evaluate f.1 we have used the linear fit to the variation of crh with wavelength determined by Koetzle and McMullan (22). Monte Carlo analysis shows that the transmission factor varies over the range of scattering angle in our experiment from 0.73 to 0.67 for H 20 and from 0.95 to 0.88 for D 20. Therefore correction for hydrogen incoherent scattering affects the relative intensities recorded within a data set as well as the overall scaling between H 20 and D 20 data sets. A De bye-Waller factor was applied to the calculated structure factor amplitudes, Fc(h). Observed structure factor amplitudes, F 0 (h). were determined by
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Figure 3-4: Views perpendicular to the helix axis of A-DNA showing density displayed at a l.3 Prms threshold representing: Figure 3 (top), map Fh. Figure 4 (bottom), map Fd.
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applying an overall scaling factor, K, to y'I0 (h_) (Figure 2). At the resolution of this study ( ~ 3A), water molecules can be approximated by single scatterin~ centres whose residual scattering length is -0.17X10- 12 em for H 20 and 1.91X10- 2 em for D 20. Therefore in the initial scaling of the HzO data set the contribution from HzO was neglected and K and B determined by least squares refinement to be 0.48 and 2A2 respectively. Since the sample was not moved during H 20/D 20 exchange these same values for K and B were used to scale the D 20 data set. Fourier synthesis studies Fourier syntheses were calculated in which observed structure factor amplitudes were combined with phases, ac, for the model of A-DNA derived from x-ray diffraction ( 13). Fh and F d maps were calculated in which the amplitudes were those observed for DNA surrounded by HzO and DzO respectively (Figures 3, 4). The threshold density is 1.3 times the root-mean-square density (Prms) and in both maps DNA is clearly visible and all negative density is greater than -1.3 Prms· While the Fh map does not show density in the non-DNA region (consistent with a water molecule at this resolution being approximated by a single scattering centre of low scattering length), the Fd map does exhibit localised water as expected. The peak heights in these maps decrease in the order base, phosphate and sugar consistent with their respective scattering lengths (Table I). Fourier difference synthesis studies In order to minimise the effect of termination errors our analysis of water structure was based on Fourier difference syntheses. Two types of synthesis were calculated in which difference amplitudes were combined with model phases, the first used the difference between amplitudes F d and Fh and the second the difference between Table I Neutron Scattering Lengths The neutron scattering lengths of different scattering groups (10- 12 em). The mean scattering density of each of the DNA groups can be obtained by dividing by the DNA scattering group volumes given by Langridge eta/ (4). The scattering length of the bases differs in H 20 and D20 because of exchange of hydrogen by deuterium at substitutable positions. Scattering group
Scattering length inHp inD 20
Phosphate Sugar Guanine Adenine Thymine Cytosine Average base H 20 D 20
2.84 1.39 7.09 6.51 4.49 4.55 5.66 -0.17
2.84 1.39 10.59 8.58 5.53 6.63 7.83 1.91
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Figure 5: View parallel to the helix axis of the A-DNA unit cell showing projected densities representing the Fd-Fh map.
amplitudes Fd and those calculated from the model of A-DNA, Fe. In both, the strength of peaks representing water relative to those representing DNA depends on the scattering power of DNA surrounded by D 20 relative to that of DNA alone. In order to remove DNA peaks in the F d-F c map, the F c amplitudes were weigh ted by the ratio of the spherically averaged amplitudes / and similarly for the Fd-Fh map. The resulting two maps display the same characteristic features at a threshold density of 1.3 Prms and in both negative density is above -1.3 Prms· A projection of map F d-Fh down the helix axis is shown in Figure 5. Almost all the difference density in these maps can be described in terms of four features. Each feature consists of a family of peaks regularly associated with the DNA double helix. These features are identified as I to IV: I) Peaks occuring between 01 oxygen atoms of neighbouring residues. A view of the F d-Fh map showing density in the major groove (Figure 6) shows how the inner ring of projected density on the central molecule in Figure 5 has its origin in these chains of peaks. II) Peaks which occur between 02 oxygen atoms of successive residues. A view of density lying across the opening of the major groove (Figure 7) shows how these peaks contribute to the outer ring of projected density around the central molecule in Figure 5. III) Peaks which occur in positions between the sugar and minor groove base edges (Figure 8). These peaks contribute along with II to the outer ring of projected density around the central molecule in Figure 5.
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Figures 6-8: Views perpendicular to the helix axis direction of A-DNA showing density in map Fd-Fh corresponding to water sites I to III: Figure 6 (left page top): family I, Figure 7 (left page, bottom): family II, Figure 8 (above): family III.
IV) A core of density which runs down the helix axis in front of the bases in the major groove, represented in Figure 5 as a peak of projected density coincident with the helix axis of the central molecule.
The identification of water molecules with difference density While the real space analysis based on the Fourier difference syntheses described above allows peaks corresponding to the water structure to be identified, this approach does not readily allow the refinement of the coordinates of water molecules associated with these peaks. A single peak may correspond to more than one water molecule and at the resolution of this study it would not in general be meaningful to attempt to refine the coordinates ofindividual water molecules. We have therefore described the water distribution in terms of a water occupancy and the coordinates of a scattering centre corresponding to a spherically averaged water molecule and refined these parameters by least squares minimisation using the function L~ (Fdi - Fcwl• where Fcwi are the amplitudes for the augmented model for theM observed reflections. The overall scaling factor and thermal parameter were refined cyclically with the water parameters. Since only water strongly associated with the DNA is likely to be represented by peaks in this study, the water thermal parameters can be assumed to be similar to those of the DNA and have therefore been tied to the overall thermal parameter. This avoids the possibility of unrealistic individual thermal parameters producing large errors in
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The final structural parameters obtained after refining the 4 water sites in the helical asymmetric unit corresponding to families I, II, III and IV. Symmetry related positions may be generated from the Cartesian coordinates (XY,Z) by using 11-fold symmetry and a dyad lying along the X-axis. The conventional crystallographic residual factor, R and the weighted residual, R" (24) are given for this model and for one refmed with the helical constraints removed Feature
X(A)
Y(A)
Z(A)
occupancy
I
3.372 2.866 4.262 1.446
-4.098 -10.15 11.529 0.751
12.860 11.998 7.440 1.033
1.47 0.79 1.34 1.64
II III IV
Model Symmetry Constrained Unconstrained
R 36.4 22.7
R" 37.8 24.0
the individual occupancy parameters due to their strong correlation. On the basis of model calculations Kundrot and Richards (23) have concluded that because of this correlation, it is inappropriate for medium resolution data to vary both occupancy and temperature factors for solvent molecules in protein structure refinements. Each of the features identified as families I to IV above consist of a group of peaks approximately related to each other by the same symmetry elements that describe the DNA double helix. There are therefore 11 crystallographically non-equivalent peaks related approximately by an 11-fold screw axis in each of these families. All the density associated with these families can therefore be described by a helical asymmetric unit which contains four peaks. The refined parameters of these four peaks are given in Table II and their positions with respect to DNA illustrated schematically in Figure 9. Inclusion of the water resulted in the conventional crystallographic residual, R, falling from 50.1% to 37 .8%. Repeating the refinement with the helical constraints removed required the introduction of an additional 40 parameters for each of the families I to IV and further reduced R to 22. 7%. The final residuals did not depend significantly on the order the families of water molecules were introduced into the refinement. The model refined without helical constraints displayed a strong negative correlation between occupancy and displacement from the positions obtained in the constrained refinement and did not represent a substantial departure from the constrained model. Using the Hamilton significance test (24) with the weighted residuals, R", given in Table II we were unable to reject the symmetry constrained model even at a 50% confidence level. Removing the symmetry constraints therefore does not significantly improve the agreement of the refined model with the experimental data. The position refined for family I is consistent with a single water bridge between adjacent 01 phosphate atoms on the same strand. Howeverits occupancy of 1.5 may reflect the participation of more than one water molecule in bridging the 01 atoms
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li Figure 9: Location with respect to two successive DNA nucleotide pairs of water sites I to IV (filled circles). Potential hydrogen bond donor and acceptor sites on the bases are represented by sectors of circles and possible interactions of the water with DNA are indicated by connecting lines.
or the contribution from water molecules bridging the 01 atoms to other DNA atoms in the major groove. Family II consists of peaks lying outside the grooves and might therefore be expected to be more influenced by the packing arrangement of DNA molecules. The position of the site refined for family II is consistent with a bridge between successive 02 phosphate atoms on the same strand. Such a bridge would require at least two water molecules although the refined occupancy is only 0.8. This suggests that the water structure represented by this family is less regular or more dynamic than that in the major groove represented by family I. The site for family III is close to the oxygen of the sugar ring and the equivalent hydrogen bond acceptor sites provided by the pyrimidine 02 and purine N3 in the minor groove. However an occupancy of 1.3 suggests that more than one water molecule is represented by this family. The position and occupancy of the site refined for family IV indicates that there are on average 1.6 water molecules per nucleotide involved in hydrating the edge of base pairs in the major groove. In the A-DNA unit cell neighbouring double helices are packed so that the rnajor groove of one molecule faces the minor groove of another. Although the lattice of A-DNA is monoclinic, the double helices are arranged in an approximately hexagonal array. Since the symmetry of the A-DNA double helix is 11-fold there are departures from equivalence in the contacts formed between the
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Figure 11: An off-axis view of A-DNA showing density representing map Fd-Fh which indicates a layer of hydration in the major groove.
various nucleotides. This is reflected in our difference maps by the fact that of the eleven peaks comprising family III, eight occur in positions close to family II peaks and one occurs close to a family III peak in neighbouring DNA molecules. This proximity is reflected in the occurrence of extended peaks in these regions of the difference maps. The positional and occupancy parameters refined by helically constrained least squares were used to calculate a new Fourier difference map by combining difference amplitudes F d-F c' with phases ac', where F c' and ac' are the structure factor amplitudes and phases calculated for DNA surrounded by localised water. This map is relatively featureless except for a chain of density running along the central channel of the major groove (Figure 10) which could represent the position of cations. The corresponding difference map in which structure factor amplitudes and phases were calculated for DNA and water positions refined without helical constraints did not differ significantly and in particular contained a similar chain of density in the major groove. Evidence in support ofthis feature being identified with cations comes from the fact that it is also weakly present in map F d-F c but not in the map F d-Fh where the cations would be expected to make the same contribution to F d and Fh. These maps also provide evidence for cations lying across the major groove in the vicinity of feature II peaks.
Discussion The results presented here show that the hydration of A-DNA can be modelled by a water structure with the same 11-fold symmetry as the double helix. In total the four
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Figure 10: View perpendicular to the helix axis direction of A-DNA showing density in map Fd-Fc' running along the central channel of the major groove.
peaks associated with each nucleotide account for 5.2 water molecules per nucleotide i.e. rather more than half of the 9 water molecules whose presence is indicated by density measurements on our sample (25). An off-axis view of map F d-Fh at a low display threshold (1.1 Prms rather than 1.3 Prms) shows how the water core running down the helix axis represented by family IV together with family I peaks linking successive phosphate oxygens along the polynucleotide chains form a deep water-lined channel which may serve as a tubular cage for cations (Figure 11 ). The distance of this layer of density from the wall of the major groove is consistent with a strongly localised primary hydration shell. In contrast to this emphasis on ordered water in the major groove as an important stabilising factor in the A-form of DNA, models proposed for the stability ofthe B-form (26) are characterised by a regular network of water in the minor groove. On this basis the humidity driven transition from the B-form to the A-form can be seen as involving the disruption of the water network in the minor groove of the B-form and the establishment of a network of hydration in the major groove of the A-form. The B to A transition can therefore be seen as a competition between grooves accompanied by a more regular association of water with the phosphate groups and the formation of single and double water bridges between adjacent phosphates. Although there are possibilities of hydrogen bonds to base and sugar atoms for water molecules in the clusters represented by families II and III it is important to
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emphasise that they occupy a region of the minor groove of A-DNA which is one of the most hydrophobic of the molecule. While the sugar ring oxygen might act as an acceptor for hydrogen bonds, this interaction can be expected to be much weaker than one to a charged phosphate oxygen or the keto group of a base. This site on the DNA has therefore some of the characteristics of a hydrophobic pocket with the localisation of water in this pocket reflecting the importance of both hydrophilic and hydrophobic interactions in stabilising DNA The hydration economy hypothesis proposed by Saenger et al (15) rests on the assumption that conformations of DNA in which oxygens in successive phosphate groups along the polynucleotide chains can be linked by single water molecules are favoured by low levels of hydration. In this hypothesis the A-form is seen as a low hydration conformation which undergoes a transition to the B-form with increasing hydration. This hypothesis was proposed primarily on the basis of water arrangements observed in single crystal x-ray diffraction studies of oligonucleotides.The observations reported here for water arrangements around the A-form of DNA itself provide evidence for the occurrence of water bridges in A-type structures like those on which the hypothesis was based. However hydration economy within the same polynucleotide strand does not account for the occurrence of the D-form of DNA (13) which is also favoured by low levels of hydration but in which successive phosphates are too far apart to be linked by a single water molecule. The occurrence of the D-form can however be accounted for by seeing the hydration economy hypothesis as part of a more general water sharing scheme in which the formation of a variety of water bridges can be assumed to be associated with low hydration conformations of DNA Lip a nov et al (27) have pointed out that in the D-form water bridges can occur across the minor groove between opposite polynucleotide strands. Our neutron fibre diffraction studies of the hydration of the D-form of poly[d(AT)].poly[d(A-T)] indicate that water is localised in this narrow minor groove (19). Neutron fibre diffraction studies of the B-form of DNA are currently underway in an attempt to further clarify the hydration patterns around the principal conformations of DNA The occupancy of the primary hydration layer lining the major groove of A-DNA identified in this study is consistent with single or double water bridges being established between all the polar and charged groups in this groove when geometrically feasible and energetically favourable (28). Similar bridges have been reported in x-ray diffraction studies of A-type oligonucleotides in different crystal environments (29). Such water sharing is therefore likely to be conserved in different packing arrangements. However water structure outside the major groove at the intermolecular contact sites is less conserved between different packing environments. In this study water has been shown to cluster in chains of pockets formed by the packing of A-type double helices. Water clusters lying in intermolecular cavities have also been reported in neutron diffraction studies of protein hydration (30). The structure of A-DNA should be considered as a complete system of interacting DNA water and cation components. We are supplementing this neutron fibre diffraction study of water around the A-DNA double helix with x-ray isomorphous
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replacement studies using synchrotron radiation to locate the cations. Such an approach emphasises the complementary nature of x-rays and neutrons as structural probes of biomolecular structure.
Acknowledgements We thank the SERC for support (Grant No. GR/F/83501), the ILL for provision of facilities, Messrs M. Daniels, G. Dudley, E. Greasley, G. Marsh, M. Wallace andJ. Archer for technical support and Mrs H.Moors for help with preparation of the manuscript References and Footnotes
1. Franklin, R.E. and Gosling, R.G.,Acta Cryst. 6, 673-677 (1953). 2. Cooper, P.J. and Hamilton, L.D.,J Mol. Bioi. 16, 562-563 (1966). 3. Fuller, W., Wilkins, M.H.F., Wilson, H.R., Hamilton, L.D. and Arnott, S.,J. Mol. Bioi. 12,60-80 (1965). 4. Langridge, R., Marvin, D.A, Seeds, W.E., Wilson, HR., Hooper, C.W., Wilkins, M.H.F. and Hamilton, L.D.,J. Mol. Bioi. 2, 38-64 (1960). 5. Marvin, D.A, Spencer, M., Wilkins, M.H.F. and Hamilton, L.D.,J. Mol. Bioi. 3, 547-565 (1961). 6. Davies, D.R., Baldwin, R.L.,J Mol. Bioi. 6, 251-255 (1963). 7. Arnott, S., Chandrasekaran, R., Birdsall, D.L., Leslie, AG.W and Ratliff, R.L., Nature 283, 743745 (1980). 8. Wang, A.H.-J, Quigley, G.J., Kolpak, F.J., Crawford, J.L., van Boom, J.H. van der Marel, G. and Rich, A, Nature 282, 680-686 (1979). 9. Mahendrasingam, A., Pigram, W.J., Fuller, W., Brahms, J. and Vergne, J.,J. Mol. Bioi. 168, 897901 (1983). 10. Mahendrasingam, A., Forsyth, V.T., Hussian, R., Greenan. R.J., Pigram, W.J., and Fuller, W., Science 233, 195-197 (1986).
11. Forsyth, V.T., Greenan, R.J., Hussian, R., Mahendrasingam, A, Nave, C., Pigram, W.J. and Fuller, W, Biochem. Soc. Trans. 14, 553-557 ( 1986). 12. Fuller, W., Forsyth, V.T. and Mahendrasingam, A, "Synchrotron Radiation and Biophysics" (Ed S.S.Hasnain), Ellis Horwood, 201-222 (1990). 13. Chandrasekaran, R. and Arnott, S.,Landolt-Bomstein New Series VII/1 b, (Ed W.Saenger), SpringerVerlag, Berlin, 38-39 & 77-78 (1989). 14. Conner, B.N., Takano, T., Tanaka, S., ltakura, K and Dickerson, R.E., Nature 295,292-299 (1982). 15. Saenger, W., Hunter, W.N. and Kennard, 0., Nature 324, 385-388 (1986). 16. Fuller, W., Mahendrasingam, A. and Forsyth, V.T., Nature 335, 596 (1988). 17. Saenger, W., Hunter, W.N. and Kennard, O.,Nature 335, 596 (1988). 18. Fuller, W., Forsyth, V.T., Mahendrasingam, A, Pigram, W.J., Greenan, R.J., Langan, P., Bellamy, K, Al-Hayalee, Y. and Mason, S.A, Physica B. 156 & 157 468-470 (1989). 19. Forsyth, V.T., Mahendrasingam, A., Pigram, W.J., Greenan, R.J., Bellamy, K, Fuller, W. and Mason, S.A,/nt. J. Macromol. 11,236-240 (1989). 20. Forsyth, V.T., Langan, P., Mahendrasingam,A. Mason, S.A and Fuller, W., Conference Proceedings of the Italian Physical Society: Water-Biomolecule Interactions, Sicily, in press (1992).
21. Rhodes, N.J., Mahendrasingam, A., Pigram, W.J., Fuller, W., Brahms, J., Vergne, J. and Warren, R.AJ, Nature 296, 267-269 (1982). 22. Koetzle, T.F. and McMullan, R.K, Research memo C4, Brookhaven National Laboratory (1980). 23. Kundrot, C.E. and Richards, F.M.,Acta Cryst. B43, 544-547 (1987). 24. Hamilton, W.C.,Acta Cryst. 18, 502-510 (1965). 25. Al-Hayalee, Y, private communication. 26. Drew, H.R. and Dickerson, R.E.,J Mol. Bioi. 151,535-556 (1981). 27. Lipanov, A.A., Beglov, D.B. and Chuprina, V.P.,J Mol. Bioi. 210,399-409 (1989). 28. Vovelle, F., Elliott, R.J. and Goodfellow, J.M., Int. J. Bioi. Macromol. 11, 39-42 ( 1989). 29. Westhof, E.,Ann. Rev. Biophys. Chem.17, 125-144(1988). 30. Kossiakoff, A.A., Sintchak, M.D., Shpungin, J. and Presta, L.G., Proteins: Structure, Function and Genetics, 12,223-236 (1992). Date Received: June 23, 1992
Communicated by the Editor Wolfram Saenger
A High Angle Neutron Fibre Diffraction Study of the Hydration of the A Conformation of the DNA Double Helix P. Langan\ V.T. Forsyth\ A. Mahendrasingam\ W J. Pigram\ S.A. Mason 2 and W. Fuller1 1
Department of Physics Keele University Staffordshire, ST5 5BG
2
Institut Laue-Langevin B.P. 156 38042 Grenoble Cedex 9, France Abstract A high angle neutron fibre diffraction study of the distribution of water around the A-form of DNA has been performed using the diffractometer Dl9 at the Institut Laue-Langevin, Grenoble. These experiments have exploited the ability to replace H 20 surrounding the DNA by D 20 so that isotopic difference Fourier maps can be computed in which peaks are identified with the distribution of water in the unit cell. All peaks of significant height have been accounted for by four families of water molecules whose positions and occupancies have been determined using least squares refinement. The coordinates of the water peaks making up each family do not deviate significantly from a regular helical arrangement with the same parameters as the DNA Two of these families are of particular intererst. The first consists of water molecules in the major groove linking successive charged phosphate oxygens along the polynucleotide chains. The second is associated with bases in the major groove and forms a central core of density along the helix axis. These two families provide a layer of hydration lining the interior wall of the major groove leaving a central channel to accommodate cations. The relationship between these observations and conformational stability is discussed.
Introduction Deoxyribonucleic acid (DNA) is a double-helical polymer which has been shown by x-ray fibre diffraction to assume a number of distinct conformations depending on hydration, ionic environment, base sequence and nearest neighbour interactions ( 1, 2). These studies have provided detailed molecular models for the A, B and C forms, the three conformations commonly available to naturally occurring DNA (3-5). The synthesis of polynucleotides with repeating base pair sequences has led to the observation of novel forms such as D for poly[ d(A-T)]. poly[d(A-T)] (6) and S (or Z) for poly[d(G-C)).poly[d(G-C)) (7, 8) as well as variants of the B form such as B"
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for poly[d(G-C)].poly[d(G-C)] (9). By exploiting the availability of the high brightness Synchrotron Radiation Source at the SERC Daresbury Laboratory we have performed time-resolved x-ray fibre diffraction studies of structural transitions in DNA(I0-12). Most synthetic and naturally occurring DNAs can assume A-type conformations in fibres where the counter ion is Na +, K+, Rb +orCs+ but not Li+, at relative humidities in the range of 66%-86%. The A-form is an 11-fo1d right-handed double helix with a pitch of 28.03A, crystallising in the monoclinic space group C2 with a=21.7A, b=39.9A, c=28.03Aand ~=96.82° (13). In early fibre diffraction experiments the Aform was observed to undergo a reversible structural transition to the B-form by increasing the relative humidity from 75% to 92% (I). Although the minor groove in B-ONA is significantly narrower than the major groove both grooves have similar depths. The displacement of the base pairs in A-DNA by about SA from their position in B-ONA away from the helix axis towards the minor groove creates a shallow minor groove and a deep major groove which in projection down the helix appears as a 6A diameter hole. Information on the hydration of DNA has come from x-ray diffraction studies of short fragments of single crystal oligonucleotide duplexes. Whereas fibre diffraction studies emphasise regularity in the double helix and factors associated with cooperative behaviour, single crystal studies highlight local variation as a function of base pair sequence. Although these techniques are complementary, care must be taken in relating results from oligonucleotides to DNA because of end effects stemming from the limited length ofthe oligonucleotide and the presence of crystallising agents. In the first A-type tetramer fragment to be studied a network of water bridging the major groove was found which Conner eta/ (14) suggested might be important in stabilising the A conformation. Similar studies on oligonucleotide duplexes in the B and Z forms have revealed an apparent correlation between the degree of extension of the DNA strands and hence the separation of adjacent phosphates and the number of water molecules associated with each phosphate group. This observation led Saenger eta/ (15) to propose that DNA conformation is determined by economy in the hydration of phosphate groups. The validity of this extrapolation of observations on oligonucleotides to polymeric DNA has been discussed (16, 17). The substantially greater scattering length of 0 20 for neutrons as compared to xrays and the possibilities for H 20/D20 isotopic substitution offer important advantages for locating water around DNA by using neutron rather than x-ray diffraction. In earlier neutron fibre diffraction studies we have exploited the difference in the scattering lengths of hydrogen and deuterium to locate water around the D-form of DNA (18, 19). Hydration patterns observed in these studies are stereochemically consistent with information from x-ray fibre diffraction studies on the location of ions within the same DNA structure (20). We describe here how these Fourier difference synthesis techniques based on the isotopic substitution of Hp by Dp in fibres of DNA have allowed us to image scattering density associated with localised water around the A-DNA double helix.
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Materials and Methods
Commercially purchased E.coli DNA was purified using previously described techniques (21) and the ionic content controlled so that fibres could be drawn which at 75% relative humidity reproducibly assumed the A conformation. Diffraction data were recorded using the single crystal diffractometer D 19 at the Institut LaueLangevin, Grenoble, with a wavelength of2.42A (19). The D19 detector is banana shaped with an aperture of 64 o X4 o, mounted with its long axis vertical so that it could be rotated about a vertical axis through the specimen. In order to achieve the desired sample size for neutron fibre diffraction studies a cuboid sample 1X5X5mm was constructed from -100 fibres each -2001J diameter and -5mm long mounted on a mica sheet. The sample was housed in a cylindrical can lOcm in diameter and 18cm in length with Melinex windows of0.036mm thickness. The relative humidity was controlled by passing helium, humidified to 75% by bubbling through an appropriate saturated salt solution, through this can. Exchange of H 20 by D 20 in the sample was achieved by replacing the salt solution by one where the solvent was D 20 and was typically complete over a period of6-8 hours. H 20/D20 exchange was monitored by observing changes in the coherent and incoherent scattering. Scattering from a standard vanadium rod was measured at the start and end of each run to calibrate the detector. Data to a resolution of3A were collected as 21 partially overlapping detector frames using an angular increment of 2.5°. Software was developed to construct a composite image of the diffraction data by merging these frames after stripping off overlapping regions to minimise edge effects. A collection time of 4 hours was required in each detector position for adequate intensity statistics. The data at each position were collected in two separate scans across the diffraction image to confirm experimental reproducibility. Figure 1 shows the images remapped into flat film geometry so thatthe distribution ofintensityis readily compared with that of typical x-ray patterns. The substantial differences in the intensity of corresponding diffraction peaks when DNA is surrounded by D 20 and H 20 are illustrated by the difference image constructed by subtracting one image from the other.
Figure 1: Neutron fibre diffraction data: (a) A-DNA surrounded by HP and (c) the difference between (a) and (b).
Dp, (b) A-DNA surrounded by
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9
8
U)
Q)
6
c
_J
1
L
s
+----------------------------~Ld~------------------------
Q)
>..
(0 _]
3
2
0. 02
0. 04
0. 06
0. 08
O. 10
0. 12
D. 1~
O. 16
0. 18
D. 2D
D. 22
Reciprocal spacing (A- 1 ) Figure 2: Comparison of the observed structure factor amplitudes corresponding to DNA surrounded by Hp (full bars) and DNA surrounded by DP (open bars). Overlaid bars correspond to accidently overlapping reflections.
Scattering from the Melinex windows was allowed for by recording diffraction with the sample removed. Once the background baseline had been subtracted from the observed diffraction patterns, Bragg intensities were determined by fitting Gaussian profiles using purpose-designed software. These intensities, I0(h). were corrected for the Lorentz effect and for attenuation using a transmission factor with a mean effective linear absorption coefficient, f.l, dominated by the effective absorption cross-section ofhydrogen, crh. In order to evaluate f.1 we have used the linear fit to the variation of crh with wavelength determined by Koetzle and McMullan (22). Monte Carlo analysis shows that the transmission factor varies over the range of scattering angle in our experiment from 0.73 to 0.67 for H 20 and from 0.95 to 0.88 for D 20. Therefore correction for hydrogen incoherent scattering affects the relative intensities recorded within a data set as well as the overall scaling between H 20 and D 20 data sets. A De bye-Waller factor was applied to the calculated structure factor amplitudes, Fc(h). Observed structure factor amplitudes, F 0 (h). were determined by
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Figure 3-4: Views perpendicular to the helix axis of A-DNA showing density displayed at a l.3 Prms threshold representing: Figure 3 (top), map Fh. Figure 4 (bottom), map Fd.
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applying an overall scaling factor, K, to y'I0 (h_) (Figure 2). At the resolution of this study ( ~ 3A), water molecules can be approximated by single scatterin~ centres whose residual scattering length is -0.17X10- 12 em for H 20 and 1.91X10- 2 em for D 20. Therefore in the initial scaling of the HzO data set the contribution from HzO was neglected and K and B determined by least squares refinement to be 0.48 and 2A2 respectively. Since the sample was not moved during H 20/D 20 exchange these same values for K and B were used to scale the D 20 data set. Fourier synthesis studies Fourier syntheses were calculated in which observed structure factor amplitudes were combined with phases, ac, for the model of A-DNA derived from x-ray diffraction ( 13). Fh and F d maps were calculated in which the amplitudes were those observed for DNA surrounded by HzO and DzO respectively (Figures 3, 4). The threshold density is 1.3 times the root-mean-square density (Prms) and in both maps DNA is clearly visible and all negative density is greater than -1.3 Prms· While the Fh map does not show density in the non-DNA region (consistent with a water molecule at this resolution being approximated by a single scattering centre of low scattering length), the Fd map does exhibit localised water as expected. The peak heights in these maps decrease in the order base, phosphate and sugar consistent with their respective scattering lengths (Table I). Fourier difference synthesis studies In order to minimise the effect of termination errors our analysis of water structure was based on Fourier difference syntheses. Two types of synthesis were calculated in which difference amplitudes were combined with model phases, the first used the difference between amplitudes F d and Fh and the second the difference between Table I Neutron Scattering Lengths The neutron scattering lengths of different scattering groups (10- 12 em). The mean scattering density of each of the DNA groups can be obtained by dividing by the DNA scattering group volumes given by Langridge eta/ (4). The scattering length of the bases differs in H 20 and D20 because of exchange of hydrogen by deuterium at substitutable positions. Scattering group
Scattering length inHp inD 20
Phosphate Sugar Guanine Adenine Thymine Cytosine Average base H 20 D 20
2.84 1.39 7.09 6.51 4.49 4.55 5.66 -0.17
2.84 1.39 10.59 8.58 5.53 6.63 7.83 1.91
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Figure 5: View parallel to the helix axis of the A-DNA unit cell showing projected densities representing the Fd-Fh map.
amplitudes Fd and those calculated from the model of A-DNA, Fe. In both, the strength of peaks representing water relative to those representing DNA depends on the scattering power of DNA surrounded by D 20 relative to that of DNA alone. In order to remove DNA peaks in the F d-F c map, the F c amplitudes were weigh ted by the ratio of the spherically averaged amplitudes / and similarly for the Fd-Fh map. The resulting two maps display the same characteristic features at a threshold density of 1.3 Prms and in both negative density is above -1.3 Prms· A projection of map F d-Fh down the helix axis is shown in Figure 5. Almost all the difference density in these maps can be described in terms of four features. Each feature consists of a family of peaks regularly associated with the DNA double helix. These features are identified as I to IV: I) Peaks occuring between 01 oxygen atoms of neighbouring residues. A view of the F d-Fh map showing density in the major groove (Figure 6) shows how the inner ring of projected density on the central molecule in Figure 5 has its origin in these chains of peaks. II) Peaks which occur between 02 oxygen atoms of successive residues. A view of density lying across the opening of the major groove (Figure 7) shows how these peaks contribute to the outer ring of projected density around the central molecule in Figure 5. III) Peaks which occur in positions between the sugar and minor groove base edges (Figure 8). These peaks contribute along with II to the outer ring of projected density around the central molecule in Figure 5.
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Structure of A-DNA
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Figures 6-8: Views perpendicular to the helix axis direction of A-DNA showing density in map Fd-Fh corresponding to water sites I to III: Figure 6 (left page top): family I, Figure 7 (left page, bottom): family II, Figure 8 (above): family III.
IV) A core of density which runs down the helix axis in front of the bases in the major groove, represented in Figure 5 as a peak of projected density coincident with the helix axis of the central molecule.
The identification of water molecules with difference density While the real space analysis based on the Fourier difference syntheses described above allows peaks corresponding to the water structure to be identified, this approach does not readily allow the refinement of the coordinates of water molecules associated with these peaks. A single peak may correspond to more than one water molecule and at the resolution of this study it would not in general be meaningful to attempt to refine the coordinates ofindividual water molecules. We have therefore described the water distribution in terms of a water occupancy and the coordinates of a scattering centre corresponding to a spherically averaged water molecule and refined these parameters by least squares minimisation using the function L~ (Fdi - Fcwl• where Fcwi are the amplitudes for the augmented model for theM observed reflections. The overall scaling factor and thermal parameter were refined cyclically with the water parameters. Since only water strongly associated with the DNA is likely to be represented by peaks in this study, the water thermal parameters can be assumed to be similar to those of the DNA and have therefore been tied to the overall thermal parameter. This avoids the possibility of unrealistic individual thermal parameters producing large errors in
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498 Table II Refined Water Parameters
The final structural parameters obtained after refining the 4 water sites in the helical asymmetric unit corresponding to families I, II, III and IV. Symmetry related positions may be generated from the Cartesian coordinates (XY,Z) by using 11-fold symmetry and a dyad lying along the X-axis. The conventional crystallographic residual factor, R and the weighted residual, R" (24) are given for this model and for one refmed with the helical constraints removed Feature
X(A)
Y(A)
Z(A)
occupancy
I
3.372 2.866 4.262 1.446
-4.098 -10.15 11.529 0.751
12.860 11.998 7.440 1.033
1.47 0.79 1.34 1.64
II III IV
Model Symmetry Constrained Unconstrained
R 36.4 22.7
R" 37.8 24.0
the individual occupancy parameters due to their strong correlation. On the basis of model calculations Kundrot and Richards (23) have concluded that because of this correlation, it is inappropriate for medium resolution data to vary both occupancy and temperature factors for solvent molecules in protein structure refinements. Each of the features identified as families I to IV above consist of a group of peaks approximately related to each other by the same symmetry elements that describe the DNA double helix. There are therefore 11 crystallographically non-equivalent peaks related approximately by an 11-fold screw axis in each of these families. All the density associated with these families can therefore be described by a helical asymmetric unit which contains four peaks. The refined parameters of these four peaks are given in Table II and their positions with respect to DNA illustrated schematically in Figure 9. Inclusion of the water resulted in the conventional crystallographic residual, R, falling from 50.1% to 37 .8%. Repeating the refinement with the helical constraints removed required the introduction of an additional 40 parameters for each of the families I to IV and further reduced R to 22. 7%. The final residuals did not depend significantly on the order the families of water molecules were introduced into the refinement. The model refined without helical constraints displayed a strong negative correlation between occupancy and displacement from the positions obtained in the constrained refinement and did not represent a substantial departure from the constrained model. Using the Hamilton significance test (24) with the weighted residuals, R", given in Table II we were unable to reject the symmetry constrained model even at a 50% confidence level. Removing the symmetry constraints therefore does not significantly improve the agreement of the refined model with the experimental data. The position refined for family I is consistent with a single water bridge between adjacent 01 phosphate atoms on the same strand. Howeverits occupancy of 1.5 may reflect the participation of more than one water molecule in bridging the 01 atoms
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li Figure 9: Location with respect to two successive DNA nucleotide pairs of water sites I to IV (filled circles). Potential hydrogen bond donor and acceptor sites on the bases are represented by sectors of circles and possible interactions of the water with DNA are indicated by connecting lines.
or the contribution from water molecules bridging the 01 atoms to other DNA atoms in the major groove. Family II consists of peaks lying outside the grooves and might therefore be expected to be more influenced by the packing arrangement of DNA molecules. The position of the site refined for family II is consistent with a bridge between successive 02 phosphate atoms on the same strand. Such a bridge would require at least two water molecules although the refined occupancy is only 0.8. This suggests that the water structure represented by this family is less regular or more dynamic than that in the major groove represented by family I. The site for family III is close to the oxygen of the sugar ring and the equivalent hydrogen bond acceptor sites provided by the pyrimidine 02 and purine N3 in the minor groove. However an occupancy of 1.3 suggests that more than one water molecule is represented by this family. The position and occupancy of the site refined for family IV indicates that there are on average 1.6 water molecules per nucleotide involved in hydrating the edge of base pairs in the major groove. In the A-DNA unit cell neighbouring double helices are packed so that the rnajor groove of one molecule faces the minor groove of another. Although the lattice of A-DNA is monoclinic, the double helices are arranged in an approximately hexagonal array. Since the symmetry of the A-DNA double helix is 11-fold there are departures from equivalence in the contacts formed between the
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Figure 11: An off-axis view of A-DNA showing density representing map Fd-Fh which indicates a layer of hydration in the major groove.
various nucleotides. This is reflected in our difference maps by the fact that of the eleven peaks comprising family III, eight occur in positions close to family II peaks and one occurs close to a family III peak in neighbouring DNA molecules. This proximity is reflected in the occurrence of extended peaks in these regions of the difference maps. The positional and occupancy parameters refined by helically constrained least squares were used to calculate a new Fourier difference map by combining difference amplitudes F d-F c' with phases ac', where F c' and ac' are the structure factor amplitudes and phases calculated for DNA surrounded by localised water. This map is relatively featureless except for a chain of density running along the central channel of the major groove (Figure 10) which could represent the position of cations. The corresponding difference map in which structure factor amplitudes and phases were calculated for DNA and water positions refined without helical constraints did not differ significantly and in particular contained a similar chain of density in the major groove. Evidence in support ofthis feature being identified with cations comes from the fact that it is also weakly present in map F d-F c but not in the map F d-Fh where the cations would be expected to make the same contribution to F d and Fh. These maps also provide evidence for cations lying across the major groove in the vicinity of feature II peaks.
Discussion The results presented here show that the hydration of A-DNA can be modelled by a water structure with the same 11-fold symmetry as the double helix. In total the four
Structure of A-DNA
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Figure 10: View perpendicular to the helix axis direction of A-DNA showing density in map Fd-Fc' running along the central channel of the major groove.
peaks associated with each nucleotide account for 5.2 water molecules per nucleotide i.e. rather more than half of the 9 water molecules whose presence is indicated by density measurements on our sample (25). An off-axis view of map F d-Fh at a low display threshold (1.1 Prms rather than 1.3 Prms) shows how the water core running down the helix axis represented by family IV together with family I peaks linking successive phosphate oxygens along the polynucleotide chains form a deep water-lined channel which may serve as a tubular cage for cations (Figure 11 ). The distance of this layer of density from the wall of the major groove is consistent with a strongly localised primary hydration shell. In contrast to this emphasis on ordered water in the major groove as an important stabilising factor in the A-form of DNA, models proposed for the stability ofthe B-form (26) are characterised by a regular network of water in the minor groove. On this basis the humidity driven transition from the B-form to the A-form can be seen as involving the disruption of the water network in the minor groove of the B-form and the establishment of a network of hydration in the major groove of the A-form. The B to A transition can therefore be seen as a competition between grooves accompanied by a more regular association of water with the phosphate groups and the formation of single and double water bridges between adjacent phosphates. Although there are possibilities of hydrogen bonds to base and sugar atoms for water molecules in the clusters represented by families II and III it is important to
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emphasise that they occupy a region of the minor groove of A-DNA which is one of the most hydrophobic of the molecule. While the sugar ring oxygen might act as an acceptor for hydrogen bonds, this interaction can be expected to be much weaker than one to a charged phosphate oxygen or the keto group of a base. This site on the DNA has therefore some of the characteristics of a hydrophobic pocket with the localisation of water in this pocket reflecting the importance of both hydrophilic and hydrophobic interactions in stabilising DNA The hydration economy hypothesis proposed by Saenger et al (15) rests on the assumption that conformations of DNA in which oxygens in successive phosphate groups along the polynucleotide chains can be linked by single water molecules are favoured by low levels of hydration. In this hypothesis the A-form is seen as a low hydration conformation which undergoes a transition to the B-form with increasing hydration. This hypothesis was proposed primarily on the basis of water arrangements observed in single crystal x-ray diffraction studies of oligonucleotides.The observations reported here for water arrangements around the A-form of DNA itself provide evidence for the occurrence of water bridges in A-type structures like those on which the hypothesis was based. However hydration economy within the same polynucleotide strand does not account for the occurrence of the D-form of DNA (13) which is also favoured by low levels of hydration but in which successive phosphates are too far apart to be linked by a single water molecule. The occurrence of the D-form can however be accounted for by seeing the hydration economy hypothesis as part of a more general water sharing scheme in which the formation of a variety of water bridges can be assumed to be associated with low hydration conformations of DNA Lip a nov et al (27) have pointed out that in the D-form water bridges can occur across the minor groove between opposite polynucleotide strands. Our neutron fibre diffraction studies of the hydration of the D-form of poly[d(AT)].poly[d(A-T)] indicate that water is localised in this narrow minor groove (19). Neutron fibre diffraction studies of the B-form of DNA are currently underway in an attempt to further clarify the hydration patterns around the principal conformations of DNA The occupancy of the primary hydration layer lining the major groove of A-DNA identified in this study is consistent with single or double water bridges being established between all the polar and charged groups in this groove when geometrically feasible and energetically favourable (28). Similar bridges have been reported in x-ray diffraction studies of A-type oligonucleotides in different crystal environments (29). Such water sharing is therefore likely to be conserved in different packing arrangements. However water structure outside the major groove at the intermolecular contact sites is less conserved between different packing environments. In this study water has been shown to cluster in chains of pockets formed by the packing of A-type double helices. Water clusters lying in intermolecular cavities have also been reported in neutron diffraction studies of protein hydration (30). The structure of A-DNA should be considered as a complete system of interacting DNA water and cation components. We are supplementing this neutron fibre diffraction study of water around the A-DNA double helix with x-ray isomorphous
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replacement studies using synchrotron radiation to locate the cations. Such an approach emphasises the complementary nature of x-rays and neutrons as structural probes of biomolecular structure.
Acknowledgements We thank the SERC for support (Grant No. GR/F/83501), the ILL for provision of facilities, Messrs M. Daniels, G. Dudley, E. Greasley, G. Marsh, M. Wallace andJ. Archer for technical support and Mrs H.Moors for help with preparation of the manuscript References and Footnotes
1. Franklin, R.E. and Gosling, R.G.,Acta Cryst. 6, 673-677 (1953). 2. Cooper, P.J. and Hamilton, L.D.,J Mol. Bioi. 16, 562-563 (1966). 3. Fuller, W., Wilkins, M.H.F., Wilson, H.R., Hamilton, L.D. and Arnott, S.,J. Mol. Bioi. 12,60-80 (1965). 4. Langridge, R., Marvin, D.A, Seeds, W.E., Wilson, HR., Hooper, C.W., Wilkins, M.H.F. and Hamilton, L.D.,J. Mol. Bioi. 2, 38-64 (1960). 5. Marvin, D.A, Spencer, M., Wilkins, M.H.F. and Hamilton, L.D.,J. Mol. Bioi. 3, 547-565 (1961). 6. Davies, D.R., Baldwin, R.L.,J Mol. Bioi. 6, 251-255 (1963). 7. Arnott, S., Chandrasekaran, R., Birdsall, D.L., Leslie, AG.W and Ratliff, R.L., Nature 283, 743745 (1980). 8. Wang, A.H.-J, Quigley, G.J., Kolpak, F.J., Crawford, J.L., van Boom, J.H. van der Marel, G. and Rich, A, Nature 282, 680-686 (1979). 9. Mahendrasingam, A., Pigram, W.J., Fuller, W., Brahms, J. and Vergne, J.,J. Mol. Bioi. 168, 897901 (1983). 10. Mahendrasingam, A., Forsyth, V.T., Hussian, R., Greenan. R.J., Pigram, W.J., and Fuller, W., Science 233, 195-197 (1986).
11. Forsyth, V.T., Greenan, R.J., Hussian, R., Mahendrasingam, A, Nave, C., Pigram, W.J. and Fuller, W, Biochem. Soc. Trans. 14, 553-557 ( 1986). 12. Fuller, W., Forsyth, V.T. and Mahendrasingam, A, "Synchrotron Radiation and Biophysics" (Ed S.S.Hasnain), Ellis Horwood, 201-222 (1990). 13. Chandrasekaran, R. and Arnott, S.,Landolt-Bomstein New Series VII/1 b, (Ed W.Saenger), SpringerVerlag, Berlin, 38-39 & 77-78 (1989). 14. Conner, B.N., Takano, T., Tanaka, S., ltakura, K and Dickerson, R.E., Nature 295,292-299 (1982). 15. Saenger, W., Hunter, W.N. and Kennard, 0., Nature 324, 385-388 (1986). 16. Fuller, W., Mahendrasingam, A. and Forsyth, V.T., Nature 335, 596 (1988). 17. Saenger, W., Hunter, W.N. and Kennard, O.,Nature 335, 596 (1988). 18. Fuller, W., Forsyth, V.T., Mahendrasingam, A, Pigram, W.J., Greenan, R.J., Langan, P., Bellamy, K, Al-Hayalee, Y. and Mason, S.A, Physica B. 156 & 157 468-470 (1989). 19. Forsyth, V.T., Mahendrasingam, A., Pigram, W.J., Greenan, R.J., Bellamy, K, Fuller, W. and Mason, S.A,/nt. J. Macromol. 11,236-240 (1989). 20. Forsyth, V.T., Langan, P., Mahendrasingam,A. Mason, S.A and Fuller, W., Conference Proceedings of the Italian Physical Society: Water-Biomolecule Interactions, Sicily, in press (1992).
21. Rhodes, N.J., Mahendrasingam, A., Pigram, W.J., Fuller, W., Brahms, J., Vergne, J. and Warren, R.AJ, Nature 296, 267-269 (1982). 22. Koetzle, T.F. and McMullan, R.K, Research memo C4, Brookhaven National Laboratory (1980). 23. Kundrot, C.E. and Richards, F.M.,Acta Cryst. B43, 544-547 (1987). 24. Hamilton, W.C.,Acta Cryst. 18, 502-510 (1965). 25. Al-Hayalee, Y, private communication. 26. Drew, H.R. and Dickerson, R.E.,J Mol. Bioi. 151,535-556 (1981). 27. Lipanov, A.A., Beglov, D.B. and Chuprina, V.P.,J Mol. Bioi. 210,399-409 (1989). 28. Vovelle, F., Elliott, R.J. and Goodfellow, J.M., Int. J. Bioi. Macromol. 11, 39-42 ( 1989). 29. Westhof, E.,Ann. Rev. Biophys. Chem.17, 125-144(1988). 30. Kossiakoff, A.A., Sintchak, M.D., Shpungin, J. and Presta, L.G., Proteins: Structure, Function and Genetics, 12,223-236 (1992). Date Received: June 23, 1992
Communicated by the Editor Wolfram Saenger
