Proc. Natl. Acad. Sci. USA Vol. 82, pp. 3611-3615, June 1985 Biochemistry Crystal structure of Z-DNA without an alternating purine-pyrimidine 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 Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and tDepartment of Organic Chemistry, 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 deoxycytidine nucleosides 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 purines and pyrimidines. However, we find that cacodylate buffer (pH 7.0), 25 mM magnesium chloride, 25 d(C-G-A-T-C-G) with the cytosines brominated or methylated mM cobalt hexamine trichloride, and 25 mM calcium chlo- on C-5 crystallizes as Z-DNA. The structure reveals thymines ride. Cobalt hexamine was used as it is known to stabilize in syn and adenines 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 cell 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 polynucleotides 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 molecule, those of the brominated derivative. Because the crystal every other base 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 cytosine residues are course ofthis refinement, 88 water molecules were identified modified either by methylation 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. thymine 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 Nucleotide 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 phosphate 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, oxygen atoms are shaded with interrupted tipped 150 toward the viewer in order to show the disposition of the circles, nitrogen atoms are shaded with fine stippled circles, the bases. The adenine 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 nucleotides 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.