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Home , Adenine, Cytosine, Guanine, Purine, Pyrimidine

Proc. Natl. Acad. Sci. USA Vol. 82, pp. 3611-3615, June 1985 Crystal structure of Z-DNA without an alternating - sequence (syn-ani conformation/ring pucker/intramolecular contacts/x-ray diffraction) ANDREW H.-J. WANG*, REINHARD V. GESSNER*, Gus A. VAN DER MARELt, JACQUES H. VAN BOOMt, AND ALEXANDER RICH* *Department of , Massachusetts Institute of Technology, Cambridge, MA 02139; and tDepartment of , Gorlaeus Laboratory, University of Leiden, 2300 RA Leiden, The Netherlands Contributed by Alexander Rich, February 8, 1985

ABSTRACT In left-handed Z-DNA, consecutive nucleo- methyl were used as the starting tides along the chain alternate in the syn and anti conforma- material (13). The purity of the oligomers was found to be tions. Purine residues form the syn conformation readily and >95% as judged by HPLC analysis. The crystallization up to now all Z-DNA crystal structures have sequences of mixture contained 2.3 mM oligonucleotide, 33 mM sodium alternating and . However, we find that cacodylate buffer (pH 7.0), 25 mM chloride, 25 d(C-G-A-T-C-G) with the brominated or methylated mM cobalt hexamine trichloride, and 25 mM chlo- on C-5 crystallizes as Z-DNA. The structure reveals ride. Cobalt hexamine was used as it is known to stabilize in syn and in anti conformations. This suggests that Z-DNA formation (14, 15). Crystal formation was induced by Z-DNA may occur in sequences other than those with alternat- using vapor-phase equilibration by equilibrating with 25% ing purine-pyrimidine sequence. 2-methyl-2,4-pentanediol at room temperature. After 3 weeks crystals began to appear in highly twinned clusters with a Double-helical DNA can exist both in right-handed and left- moderate yellow color. A triangular plate was placed in a handed forms. The structure of right-handed B-DNA has been glass capillary surrounded by a droplet of mother liquor for known since 1953 and an atomic resolution single-crystal x-ray x-ray analysis. The brominated hexamer crystal was found to diffraction analysis in 1979 showed that the DNA hexamer be orthorhombic with space group P212121 and dimen- d(CpGpCpGpCpG) [(dC-dG)31 forms a left-handed helix called sions a = 18.3, b = 31.1, and c = 44.1 A. The methylated Z-DNA (1). A variety of studies carried out on oligodeoxynu- derivatives crystallized as small quasihexagonal rods with cleotide single crystals (2-6) and on has shown maximum dimensions of 0.1 mm. The space group and cell that Z-DNA can form in sequences with alternations ofpurines dimensions of the methylated derivative were very similar to and pyrimidines (see review in ref. 7). In the Z-DNA , those of the brominated derivative. Because the crystal every other adopts the syn conformation relative to the fragments of the brominated derivative were larger, they sugar, in contrast to B-DNA, where all of the bases are found were used for data collection on a Nicolet P3 diffractometer in the anti conformation. Purine nucleosides can adopt the syn out to a resolution of 1.54 A using the o-scan mode. The total conformation as easily as they can adopt the anti conformation, number of observable reflections with an intensity greater while pyrimidine nucleosides do this less readily (8, 9). How- than 1.5 o(I) was 2386. The cell dimensions of this crystal ever, we do not know whether pyrimidines can adopt the syn were similar to those that have been observed in other conformation in DNA. It has been shown that negative orthorhombic single crystals of DNA hexamer duplexes and supercoiling can stabilize Z-DNA formation in plasmids (10, so a set oftrial coordinates from an appropriate sequence was 11). Studies of supercoiled plasmids suggested that segments generated from the (dC-dG)3 crystal structure (1). The can form Z-DNA without a strict alternation of purines and Konnert-Hendrickson refinement method was used and the pyrimidines (12). Here we report that a DNA fragment with the structure was refined to a final R value of 19.3% (16). In the sequence d(CpGpApTpCpG), where the residues are course ofthis refinement, 88 were identified modified either by or bromination on the C-5 solvating the oligonucleotide fragment. Although the crystals position, crystallizes in the form of left-handed Z-DNA. Ex- had a yellow color, the cobalt hexamine could not be located amination of the crystal structure reveals that the two central unambiguously in the final Fourier map. residues adopt the syn conformation with intramolecular distances that are only slightly shorter than those The Backbone Conformation of Z-DNA Is Independent of seen with purine residues in the syn conformation. Two of the Sequence six residues in this molecule no longer maintain an alternation of purines and pyrimidines and still it forms Z-DNA. This In order to form the Z-DNA conformation in this sequence, suggests that segments of DNA may form left-handed Z-DNA we expected the two thymine residues to adopt the syn without a strict adherence to the alternation of purines and conformation. This might be accomplished through a signifi- pyrimidines. In addition, the presence of syn pyrimidines and cant modification of the Z-DNA backbone. Instead, the anti purines in Z-DNA changes the external shape of the backbone was similar to that seen in the initial (dC-dG)3 molecule. structure (1) as well as in the structures found in the d(m5C-G-T-A-m5C-G) (6). It appears that the thymine resi- Crystallization and Structure Solution dues comfortably adopted a syn conformation in the struc- ture. However, the outer surface features of the Z-DNA The oligonucleotides were synthesized by an improved molecule looked somewhat different. In Z-DNA, unlike triester method in which either 5-bromo or 5- B-DNA, the base pairs are on the outer surface of the molecule. In this molecule there are irregularities where the The publication costs of this article were defrayed in part by page charge purines are in the anti conformation and the pyrimidines in payment. This article must therefore be hereby marked "advertisement" syn. This can be readily seen in Fig. 1, which shows van der in accordance with 18 U.S.C. §1734 solely to indicate this fact. Waals diagrams of (dC-dG)3 and d(br5C-G-A-T-br5C-G).

3611 Downloaded by guest on September 26, 2021 3612 Biochemistry: Wang et al. Proc. Natl. Acad. Sci. USA 82 (1985)

d(CGCGCG) d( CGAT'CG) FIG. 1. A van der Waals diagram showing the structure of d(C-G-C-G-C-G) and of d(br5C-G-A-T-br5C-G). The molecules are shown just as they appear in the crystal lattice with three molecules stacked upon each other along the c axis. There is a continuity ofbase stacking although every sixth phosphate group is missing along the backbone. A solid line going from phosphate to phosphate group illustrates the zigzag nature of the nucleotide backbone and the groove between them is shaded. The arrows point to the protruding thymine groups in the syn conformation; otherwise, the similarity of FIG. 2. A stereo diagram of the structure of d(br5C-G-A-T-br5C- the two molecules is evident. The phosphorus atoms are drawn as G). Two molecules are seen in this diagram and the helical axis is dotted concentric circles, atoms are shaded with interrupted tipped 150 toward the viewer in order to show the disposition of the circles, atoms are shaded with fine stippled circles, the bases. The residues in the anti position are very close to the bromine atom is drawn with solid circles, and carbon atoms are helical axis, while the thymine residues in the syn position are drawn with smaller concentric circles. considerably removed. The COG base pairs are largely coplanar, but there are 11° and 120 dihedral angles between the planes of the These each show three molecules of the double-helical adenine and thymine residues. hexamer as they are found in the crystal lattice. Both structures have a continuity ofbase stacking, so the molecule molecule is similar to that seen in the (dC-dG)3 structure. appears as ifit formed a continuous double helix even though Sugar rings along the chain have their 0-1' atoms oriented every sixth phosphate group is missing as the molecules are alternately either up or down as seen in all Z-DNA structures. hexanucleoside pentaphosphates. A major difference be- tween the two structures is illustrated by the thymine Changes in Base Stacking residues in the syn conformation that project away from the axis of the molecule (indicated by arrows in Fig. 1). The In B-DNA the asymmetric unit is one nucleotide and the adenine residues in the anti conformation are positioned stacking between adjacent base pairs is somewhat similar, as somewhat closer to the axis of the molecule producing a they lie over the helix axis ofthe molecule. Because there are surface indentation. The change in the conformation of these two in the asymmetric unit of Z-DNA, there are two bases thus produces a significant irregularity in the two different types of stacking interactions (7, 17). In the external contour of the DNA due to changes in base se- (dC-dG)3 crystal structure, CpG sequences were found to be quence. This is quite at variance with B-DNA in which largely sheared with the cytosines stacked over the cytosines changes in nucleotide sequence produce little modification in of the opposite strands and a rotation of only -9° between the external form of the molecule. There is also a significant adjacent base pairs. In contrast to this, the GpC sequences change in the distribution of electronegative nitrogen and had stacking between the bases on the same strand and a large oxygen atoms on the Z-DNA surface with syn pyrimidines rotation of -51° between each (1). and anti purines. Fig. 3 shows the stacking of successive base pairs in the A more detailed view is shown in the stereo diagram ofFig. molecule d(br5C-G-A-T-br5C-G). The nucleotides in the mol- 2 in which the molecule is tilted 150 toward the reader so that ecule are numbered 1-6 along one strand and 7-12 along the the bases can all be seen. It is clear that the thymines are in other, so that br5Cl of one strand is bonded to G12 the syn conformation and adenines in anti. The adenine of the other. In Fig. 3a the base pair br5C1 G12 (solid lines) residues are closer to the axis and the extent to which the is shown projected over the base pair G2 br5C11 (open lines). thymine residues protrude can also be seen. Although the The sequence ofthe molecules on the left strand is br5ClpG2. C-G base pairs are largely coplanar, the two A-T base pairs All of the molecules in the top row (Fig. 3a-c) have the have dihedral angles of 110 and 120. It is not clear whether this conformation anti-p-syn for the dinucleotide sequence. This is intrinsic in the molecule or is a perturbation due to the is in contrast to the second row (Fig. 3 d-f) in which the bromine atom. The polarizable bromine atoms are seen to molecules have the sequence syn-p-anti in going from the 5' stack over the thymine rings and this may induce a slight to 3' direction. The essential elements of the Z-DNA struc- change in their orientation. The backbone of this Z-DNA ture are represented by the alternation of confor- Downloaded by guest on September 26, 2021 Biochemistry: Wang et al. Proc. Natl. Acad. Sci. USA 82 (1985) 3613

a b

(3--- T10

d

FIG. 3. Successive base pairs are viewed down the helix axis in order to show their stacking interactions. The base pair in solid lines is closer to the reader than the base pair drawn with open lines and the solid dot is the helical axis. The base pairs are identified by the symbols under them, where C stands for br5C. Thus, in a the base pair br5C1 G12 lies above the base pair G2-br5C11. (a-c) Sequences in which the conformation is anti-p-syn, which means that the anti residue is on the 5' side of the phosphate and the residue in the syn conformation on the 3' side. (d-f) Sequences in which conformations are syn-p-anti. (f) The base pair G6 br5C7 as it stacks upon the base pair br5C1' G12' of the molecule below.

mations along the nucleotide chain rather than by the se- to Fig. 3f. The cytosine and thymine residues do not stack quence of particular nucleotides. upon each other directly but T4 lies on top of the bromine The three anti-p-syn sequences in the top row of Fig. 3 all atom of the C-5 residue (Fig. 3e), and the bromine atom of show a sheared orientation of the base pairs with some C-li overlays T10 (Fig. 3d). Similarly, there is little stacking stacking in the center from opposite strands as described for seen between the two purines G2 and A3 (Fig. 3d) and A9 and the (dC-dG)3 structure (1). These sequences pre also associ- G8 (Fig. 3e). There is significantly less base stacking in the ated with small changes in the twist angle. However, there syn-p-anti sequences. The twist angles of all ofthe sequences are some significant differences from the (dC-dG)3 due to the are very similar to each other, averaging -49° introduction of thymine residues in syn and adenine residues in anti conformation. Fig. 3 a and c show stacking inter- syn Conformation of Pyrimidine Nucleotides actions that are similar to those seen in the (dC-dG)3 structure; the CpG base pairs are sheared so that the All of the nucleotides in right-handed B-DNA are in the anti residues stack over each other slightly in the center. The conformation while in Z-DNA every other base is in the syn residues do not stack on bases but rather on the 0-1' conformation (1). Purines are believed to form the syn atom of the furanose ring below them. However, in Fig. 3b, conformation with little strain and this fits with the observa- the two adenine residues in the anti conformation are found tion that Z-DNA formation is favored by sequences with to stack directly on top of each other with a greater stacking alternations of purines and pyrimidines (7). The present interaction than that seen for the two central cytosine structure shows that a Z-DNA conformation can form even residues in Fig. 3 a and c, while the thymine residues in Fig. if the nucleotide sequences are out of alternation. The 3b are found to have very little stacking on one face. They are conformation of several nucleotides in this structure is shown in weak van der Waals contact with the adjacent nucleotide, in Fig. 4, which illustrates the orientation of the furanose ring as the distance between the C-5' of and the relative to the base. The column on the left has syn nucleo- thymine ring is near 3.5 A. It can also be seen that the bulk tides, including the two thymine residues, while the column of the adenine ring is significantly closer to the axis of the on the right shows anti conformations, including the two molecule (solid dot) in Fig. 3b than are the cytosine rings in adenine nucleotides. In the anti conformation, the atoms of Fig. 3 a and c, but the thymine rings are further away from the furanose ring are rotated so that they are no longer in the axis. The distances from the axis to the methyl group of contact with the atoms of the base. However, in the syn the thymines T4 and T7 are 8.7 and 9.3 A, respectively. These conformation there are a number of "back" contacts- atoms are the furthest removed from the axis of any base namely, contacts between atoms of the furanose ring and the atom in the molecule. In the (dC-dG)3 structure, the furthest base. As seen in Fig. 4, the close contacts involve 0-2 of base atom was guanine C-8, which is 7.8 A away from the axis pyrimidines T4 and T10 as well as N-3 of the purines in the (15). However, if a cytidine residue were in the syn conforma- syn conformation. These contacts are listed in Table 1 for all tion, its apparent displacement would be less because of the of the nucleotides in the syn conformation. absence of the methyl group. The twist angles for the two It is of interest to compare the intramolecular contacts base pairs in Fig. 3 a and c are -13° and -12°, respectively, between the syn pyrimidine nucleosides T4 and T10 with the while for Fig. 3b it is just under -9° four syn guanine nucleosides in Table 1. The distances The lower row in Fig. 3 shows the stacking ofthe base pairs between 0-2 and the furanose 0-1' are slightly longer than the in the syn-p-anti sequence. Fig. 3f shows the stacking of the van der Waals distance of 2.8 A. However, the contacts bottom C-G base pair of one hexamer molecule with the upper between 0-2 and C-2' or C-3' in the pyrimidines are slightly G-C base pair of the hexamer molecule immediately below. shorter than those between N-3 and C-2' and C-3' for the The stacking of the bases at this position between the two purines. The shortest distance (2.6 A) is found between molecules is indistinguishable from the stacking of the GpC thymine 0-2 and C-2' of T10 and distances similar to that residues within (4C-dG)3. However, significant differences have been seen in pyrimidine nucleosides that have crystal- are seen in the sequences involving the A-T base pairs. In Fig. lized in the syn conformation (18, 19). These contacts are 3 d and e, the stacking is shown for segments in which two sensitive to slight changes in the rotation of the base about its pyrimidines are on one strand and two purines on the other. glycosyl bond. As mentioned above, the thymine rings are in There is somewhat less stacking ofbases over each other than direct van der Waals contact with bromine atoms on the is found in the GpC sequences of (dC-dG)3, which is similar adjacent nucleotide and it is possible these might perturb the Downloaded by guest on September 26, 2021 3614 Biochemistry: Wang et al. Proc. Natl. Acad. Sci. USA 82 (1985)

Table 2. Sugar pucker and X angles in d(br5C-G-A-T-br5C-G) Pucker Residue By distance Type X, degrees anti br5C1 C-3' exo C-2' endo -136 T4, syn, x=59' A9, anti, x=-15W A3 C-2' endo C-2' endo -137 br5C5 C-1' exo C-2' endo -178 br5C7 C-2' endo C-2' endo -152 A9 C-2' endo C-2' endo -150 br5ClW C-1' exo C-2' endo -163 syn G2 C-3' endo C-3' endo 59 T4 C-4' exo C-3' endo 59 G6 C-3' exo C-2' endo 78 T10, syn, X=75' A3, anti, X=-137- G8 C-4' exo C-3' endo 64 T10 C-3' endo C-3' endo 75 G12 C-2' endo C-2' endo 84 The sugar puckers are listed by the maximum distance of an individual atom from the plane of the four remaining atoms as well as by the type of sugar pucker-i.e., C-2' endo or C-3' endo.

G8, syn, X=64' C5, anti, x=-178' DISCUSSION Z-DNA is a higher energy form of the double helix (7). The FIG. 4. The position of the ring is shown relative to conversion of right-handed B-DNA to left-handed Z-DNA is the bases for a number of residues. The bases are drawn as if they accompanied by rotation about the glycosyl bonds of every were lying on a horizontal surface and the sugars are seen to adopt different positions around the glycosyl bond that is in the plane ofthe other residue from an anti to a syn conformation. Since bases but extending away from the reader. All of the residues on the purine residues can adopt the syn conformation more readily left are in the syn conformation and those on the right are in the anti than pyrimidines (8), Z-DNA formation is favored in se- conformation and the angle of rotation (X) is indicated. The closest quences with alternations of purines and pyrimidines. How- contacts between the base and the sugar are found in the syn ever, we find that a molecule without an alternating sequence conformations between 0-2 of pyrimidines or N-3 of purines and the appears to be able to crystallize as Z-DNA. An important various atoms 0-1', C-2', and C-3' of the sugar ring. point is how much additional energy is required for this process. If the energy difference is small, then such se- glycosyl angle. However, the important general observation quences may form Z-DNA readily with a number ofbases out that the intramolecular contacts for the thymine residues in of purine pyrimidine alternation. Until recently there have is been no measurements for estimating the likelihood of the syn conformation are only slightly shorter than those forming Z-DNA in nucleotide sequences that do not alternate found for the guanine residues in the syn conformation. in purines and pyrimidines. The present structure allows us The base to sugar "back contacts" are sensitive to the to make some qualitative estimates. pucker of the furanose rings. In general, adoption of a C-3' Two factors stabilize the higher energy Z-DNA in biologi- endo conformation in the syn nucleotides removes unfavor- cal systems. In negatively supercoiled DNA, the energy of able short contacts. The sugar pucker and the glycosyl negative supercoiling is used to stabilize Z-DNA formation torsional angles are listed in Table 2 for all of the residues. It (7). Likewise, Z-DNA binding can stabilize the can be seen that nucleosides T4 and T10 both adopt puckers Z-DNA conformation in a complex (7). In experiments that are the C-3' endo type, while the adenine residues in the carried out with negatively supercoiled plasmid pBR322, a anti conformation A3 and A9 both adopt puckers of the C-2' 14-base-pair sequence of alternating purines and pyrimidines endo type. In the original (dC-dG)3 Z-DNA structure, all of with 1 base pair out of alternation was identified as a site of the cytosine residues were anti and had the C-2' endo Z-DNA formation (12). In the present work, a chemically conformation, while the guanine residues were all syn and modified hexanucleotide with 2 of 6 base pairs out of were found in the C-3' endo conformation (except for end purine-pyrimidine alternation forms Z-DNA in a single effects). However, all of the values fall in a fairly tight range, crystal lattice. with a distribution of angles encompassing <300 in the syn The present structure shows that the backbone can ac- group and only a slightly greater spread for the anti conforma- commodate Z-DNA formation in a nonalternating purine- tions. pyrimidine sequence. The root-mean-square (rms) deviation of the backbone of the present structure compared to the (dC-dG)3 structure is only 0.45 A. This is in contrast to the Table 1. Close contacts of nucleotides in the syn conformation: bases that deviate significantly in their position in the Distances between the furanose atoms and guanine N-3 or out-of-alternation segments. thymine 0-2 This structure raises a number of questions. For example, Base Atom 0-1' C-2' C-3' there is a stabilizing stacking of adenine A3 on adenine A9 and to what extent does this interaction compensate for the G2 N-3 3.1 3.4 3.2 is the T4 0-2 3.0 3.0 3.1 loss of stacking of thymine residues? Also, what G6 N-3 3.5 3.2 4.1 relative energetic contribution of one base pair out ofalterna- G8 N-3 3.4 3.0 3.3 tion versus two? Finally, do AT base pairs form out-of- T10 0-2 3.2 2.6 3.2 alternation Z-DNA structure more easily than do O0C base G12 N-3 3.6 3.2 4.4 pairs? In this structure there is a modification in the external shape of the molecule in the region that is out of Downloaded by guest on September 26, 2021 Biochemistry: Wang et al. Proc. Natl. Acad. Sci. USA 82 (1985) 3615 purine-pyrimidine alternation. Such sequence-dependent might occur more widely, including some sequences that shape changes are not seen in B-DNA. An irregular shape on contain base pairs out of purine-pyrimidine alternation. This the surface of Z-DNA suggests the possibility that this locus makes it more difficult to recognize potential Z-DNA seg- might be easily recognized, for example, by a or other ments by simply scanning nucleotide sequences. Nonethe- molecule binding to its surface. less, alternations ofpurines and pyrimidines are still the most Previously, it had been shown that there are two conforma- likely for forming Z-DNA. We may now define Z-DNA as a tions of phosphate groups in Z-DNA-namely, Zj and Z11 left-handed double helix held together by Watson-Crick base (20). In the present structure, the phosphate P-5 is in the Z11 pairs in which the bases alternate in anti and syn conforma- conformation and the other nine are all in the Z1 conforma- tions. This definition of Z-DNA can be applied to any tion. The P-5 phosphate has the Z11 conformation because nucleotide sequence that may form Z-DNA. The extent to there is a magnesium ion complexed to the N-7 of guanine-6 which significant segments of Z-DNA form with base pairs in a manner similar to that seen in the (dC-dG)3 structure (20). out of purine-pyrimidine alternation remains to be deter- In the (dC-dG)3 structure, the water molecules are highly mined experimentally. organized in the deep helical groove. The Z-DNA crystal structure of the hexanucleotide d(C-G-T-A-C-G) has been This research was supported by grants from the National Institutes reported in which the cytosine residues are modified by of Health, the American Society, National Aeronautics and methylation or bromination ip the C-5 position (6). The water Space Administration, the Office of Naval Research, the National Science Foundation, as well as the Netherlands Organization for the molecules in the helical groove are disordered near the ANT Advancement of Pure Research (ZWO). R.V.G. was supported by a base pairs. In the present structure the organization of the grant from Studienstiftung des Deutschen Volkes. water is different from that seen in (dC-dG)3. Several of the water molecules are now found in the plane of the base pairs 1. Wang, A. H.-J., Quigley, G. J., Kolpak, F. J., Crawford, that, in the structures above, were found between the base J. L., van Boom, J. H., van der Marel, G. & Rich, A. (1979) pairs. This may reflect the fact that the A-T base pairs have Nature (London) 282, 680-686. adenine N-3 near the axis of the molecule and, unlike the 0-2 2. Drew, H., Takano, T., Tanaka, S., Itakura, K. & Dickerson, of pyrimidines, adenine N-3 favors hydrogen bonding to R. E. (1980) Nature (London) 286, 567-573. water in the plane of the base. No large segments of 3. Crawford, J. L., Kolpak, F. J., Wang, A. H.-J., Quigley, disordered water molecules were found in the groove of the G. J., van Boom, J. H., van der Marel, G. & Rich, A. (1980) Proc. Natl. Acad. Sci. USA 77, 4016-4020. present structure. 4. Drew, H. R. & Dickerson, R. E. (1981) J. Mol. Biol. 152, A few crystallographic studies have been carried out of 723-736. pyrimidine residues that crystallize in the syn conformation 5. Fujii, S., Wang, A. H.-J., van der Marel, G., van Boom, J. H. (18) (see review in ref. 19). They are usually associated with & Rich, A. (1982) Nucleic Acids Res. 10, 7879-7892. some modification of the pyrimidine that favors the syn 6. Wang, A. H.-J., Hakoshima, T., van der Marel, G., van conformation. For example, substitution of a cyano group on Boom, J. H. & Rich, A. (1984) Cell 37, 321-331. the C-6 position of yields a molecule that crystallizes 7. Rich, A., Nordheim, A. & Wang, A. H.-J. (1984) Annu. Rev. in the syn conformation (19). The distances between the 0-2 Biochem. 53, 791-846. and the furanose ring atoms in that modified 6-cyanouridine 8. Haschemeyer, A. E. V. & Rich, A. (1%7) J. Mol. Biol. 27, As 369-384. structure are similar to those seen in the present structure. 9. Davies, D. B. (1978) Progress in NMR in most of the pyrimidines that crystallize in the syn confor- (Pergamon, Oxford), Vol. 12, pp. 135-186. mation, the pucker of the furanose rings is suitably adjusted 10. Singleton, C. K., Klysik, J., Stirdivant, S. M. & Wells, R. D. to minimize close contacts with the base. (1982) Nature (London) 299, 312-316. In the present structure, Z-DNA stabilization has been 11. Peck, L. J., Nordheim, A., Rich, A. & Wang, J. C. (1982) enhanced by including either methyl groups or bromine Proc. Natl. Acad. Sci. USA 79, 45604564. atoms on the 5 position of cytosine. In addition, cobalt 12. Nordheim, A., Lafer, E. M., Peck, L. J., Wang, J. C., Stollar, hexamine has been included in the crystallization mixture. B. D. & Rich, A. (1982) Cell 31, 309-318. We have previously reported the structure of the cobalt 13. van der Marel, G., van Boeckel, C. A. A., Wille, G. & van hexamine complex of (dC-dG)3 (15). Cobalt hexamine stabi- Boom, J. H. (1981) Tetrahedron Lett. 22, 3887. lizes that molecule by making 5 hydrogen bonds to the outer 14. Behe, M. & Felsenfeld, G. (1981) Proc. Natl. Acad. Sci. USA part of the molecule to the guanine residue and a neighboring 78, 1619-1623. phosphate. The positions occupied by cobalt hexamine in the 15. Gessner, R., Quigley, G. J., Wang, A. H.-J., van der Marel, available in the G., van Boom, J. H. & Rich, A. (1984) Biochemistry 24, (dC-dG)3 crystal lattices are no longer present 237-240. structure since they are now occupied by the A-T base pairs. 16. Hendrickson, W. A. & Konnert, J. (1979) in Biomolecular Although the crystals are yellow no cobalt hexamine was Structure, Conformation, Function and , ed. identified in the lattice, probably due to disordering. How- Srinivasan, R. (Pergamon, Oxford), pp. 43-57. ever, crystallization is not essential since studies of an 17. Wang, A. H.-J., Fujii, S., van Boom, J. H. & Rich, A. (1983) oligonucleotide without purine-pyrimidine alternation yield Cold Spring Harbor Symp. Quant. Biol. 47, 33-44. NMR data demonstrating Z-DNA with syn thymines in 18. Saenger, W. & Scheit, K. H. (1970) J. Mol. Biol. 50, 153-169. solution. 19. Yamagata, Y., Kobayashi, Y., Okabe, N., Tomita, K., Sano, At present, the potential for Z-DNA formation has been T., Inoue, H. & Ueda, T. (1983) Nucleosides Nucleotides 2, sought in DNA sequences by searching out segments that 335-343. have alternations of purines and pyrimidines. Crystallization 20. Wang, A. H.-J., Quigley, G. J., Kolpak, F. J., van der Marel, of this out-of-alternation sequence suggests that Z-DNA G., van Boom, J. H. & Rich, A. (1981) Science 211, 171-176. Downloaded by guest on September 26, 2021