Crystal structure of a B-DNA dodecamer containing inosine, d(CGCIAATTCGCG), at 2.4 A resolution and its comparison with other B-DNA dodecamers.

The crystal structure of the dodecamer, d(CGCIAATTCGCG), has been determined at 2.4 A resolution by molecular replacement, and refined to an R-factor of 0.174. The structure is isomorphous with that of the B-DNA dodecamer, d(CGCGAATTCGCG), in space group P2(1)2(1)2(1) with cell dimensions of a = 24.9, b = 40.4, and c = 66.4 A. The initial difference Fourier maps clearly indicated the presence of inosine instead of guanine. The structure was refined with 44 water molecules, and compared to the parent dodecamer. Overall the two structures are very similar, and the I:C forms Watson-Crick base pairs with similar hydrogen bond geometry to the G:C base pairs. The propeller twist angle is low for I4:C21 and relatively high for the I16:C9 base pair (-3.2 degrees compared to -23.0 degrees), and the buckle angles alter, probably due to differences in the contacts with symmetry related molecules in the crystal lattice. The central base pairs of d(CGCIAATTCGCG) show the large propeller twist angles, and the narrow minor groove that characterize A-tract DNA, although I:C base pairs cannot form the major groove bifurcated hydrogen bonds that are possible for A:T base pairs.     

Crystal structure of the self-complementary 5''-purine start decamer d(GCACGCGTGC) in the A-DNA conformation. II.

The crystal structure of the alternating 5'-purine start decamer d(GCGCGCGCGC) was found to be in the left-handed Z-DNA conformation. Inasmuch as the A.T base pair is known to resist Z-DNA formation, we substituted A.T base pairs in the dyad-related positions of the decamer duplex. The alternating self-complementary decamer d(GCACGCGTGC) crystallizes in a different hexagonal space group, P6(1)22, with very different unit cell dimensions a = b = 38.97 and c = 77.34 A compared with the all-G.C alternating decamer. The A.T-containing decamer has one strand in the asymmetric unit, and because it is isomorphous to some other A-DNA decamers it was considered also to be right-handed. The structure was refined, starting with the atomic coordinates of the A-DNA decamer d(GCGGGCCCGC), by use of 2491 unique reflections out to 1.9-A resolution. The refinement converged to an R value of 18.6% for a total of 202 nucleotide atoms and 32 water molecules. This research further demonstrates that A.T base pairs not only resist the formation of Z-DNA but can also assist the formation of A-DNA by switching the helix handedness when the oligomer starts with a 5'-purine; also, the length of the inner Z-DNA stretch (d(CG)n) is reduced from an octamer to a tetramer. It may be noted that these oligonucleotide properties are in crystals and not necessarily in solutions.     

Crystal structure of d(GCGCGCG) with 5''-overhang G residues.

The crystal structure of the DNA heptamer d(GCGCGCG) has been solved at 1.65 A resolution by the molecular replacement method and refined to an R-value of 0.184 for 3598 reflections. The heptamer forms a Z-DNA d(CGCGCG)2 with 5'-overhang G residues instead of an A-DNA d(GCGCGC)2 with 3'-overhang G residues. The overhang G residues from parallel strands of two adjacent duplexes form a trans reverse Hoogsteen G x G basepair that stacks on the six Z-DNA basepairs to produce a pseudocontinuous helix. The reverse Hoogsteen G x G basepair is unusual in that the displacement of one G base relative to the other allows them to participate in a bifurcated (G1)N2 . . . N7(G8) and an enhanced (G8)C8-H . . . O6(G1) hydrogen bond, in addition to the two usual hydrogen bonds. The 5'-overhang G residues are anti and C2'-endo while the 3'-terminal G residues are syn and C2'-endo. The conformations of both G residues are different from the syn/C3'-endo for the guanosine in a standard Z-DNA. The two cobalt hexammine ions bind to the phosphate groups in both GpC and CpG steps in Z(I) and Z(II) conformations. The water structure motif is similar to the other Z-DNA structures.     

Hexagonal crystal structure of the A-DNA octamer d(GTGTACAC) and its comparison with the tetragonal structure: correlated variations in helical parameters

The alternating DNA octamer d(GTGTACAC) has been grown in a novel hexagonal crystal form. The structure has been determined and refined to a 2-A resolution, with 51 water molecules. The A-DNA conformation is a variant of that observed for the tetragonal form of the same sequence (Jain et al., 1989) containing a bound spermine. The crystals belong to the space group P6(1)22, a = b = 32.40 A and c = 79.25 A, with one strand in the asymmetric unit. The new hexagonal structure was solved by rotation and translation searches in direct space and refined to a final R value of 12.7% by using 1561 unique reflections greater than 1.5 sigma (I). The electron density clearly shows that the penultimate A7 sugar had flipped into the alternative C2'-endo pucker. This dent in the molecule can be attributed to close intermolecular contacts. In contrast, in the tetragonal structure, the DNA is distorted in the central TA step, where the A5 backbone bonds C4'-C5' and O5'-P assume trans conformations. The hexagonal double helix more closely resembles the fiber diffraction A-DNA, compared to the tetragonal form. For instance, the tilt angle is higher (16 degrees vs 10 degrees), which is correlated with a larger displacement from the helix axis (3.5 vs 3.3), a lower rise per residue (2.9 vs 3.2), and a smaller major-groove width (6.1 vs 8.7), thus indicating that the variations in these global helical parameters are correlated. The propeller twist angles in both forms are higher for the G-C base pairs (15.3 degrees, 12.14 degrees) than for the A-T base pairs (10.8 degrees, 9.1 degrees), which is the reverse of the expected order. Unlike the tetragonal structure, the hexagonal crystal structure interestingly does not contain a bound spermine molecule. Our analysis reveals that the conformational differences between the tetragonal and hexagonal forms are not entirely due to the spermine binding, and crystal packing seems to play an important role.     

Crystallization and preliminary crystallographic studies of the decadeoxyoligonucleotide d(CpGpTpApCpGpTpApCpG)

Crystals of the self-complementary decadeoxyoligonucleotide d(CpGpTpApCpGpTpApCpG) have been grown from a solution containing [Co(NH3)6]Cl3 and spermine. The amber-colored crystals are hexagonal and belong to the space group P6(5) (or P6(1] with unit cell parameters a = 17.93 A, c = 43.41 A. Precession photography and molecular packing considerations indicate that the unit cell consists of a 12 nucleotide duplex. The asymmetric unit, therefore, is a disordered duplex dimer in which each pyrimidine-purine base-pair is occupied 60% of the time by a C . G pair and 40% of the time by a T . A pair. The above considerations and preliminary structure analysis reveal that this alternating pyrimidine-purine oligomer assumes a Z-DNA conformation.     

Crystal structure of a Z-DNA hexamer d(CGCICG) at 1.7 A resolution: inosine.cytidine base-pairing, and comparison with other Z-DNA structures.

The crystal structure of the deoxyhexamer, d(CGCICG), has been determined and refined to a resolution of 1.7A. The DNA hexamer crystallises in space group P2(1)2(1)2(1) with unit cell dimensions of a = 18.412 +/- .017 A, b = 30.485 +/- .036A, and c = 43.318 +/- .024 A. The structure has been solved by rotation and translation searches and refined to an R-factor of 0.148 using 2678 unique reflections greater than 1.0 sigma (F) between 10.0-1.7 A resolution. Although the crystal parameters are similar to several previously reported Z-DNA hexamers, this inosine containing Z-DNA differs in the relative orientation, position, and crystal packing interactions compared to d(CGCGCG) DNA. Many of these differences in the inosine form of Z-DNA can be explained by crystal packing interactions, which are responsible for distortions of the duplex at different locations. The most noteworthy features of the inosine form of Z-DNA as a result of such distortions are: (1) sugar puckers for the inosines are of C4'-exo type, (2) all phosphates have the Zl conformation, and (3) narrower minor grove and compression along the helical axis compared to d(CGCGCG) DNA. In addition, the substitution of guanosine by inosine appears to have resulted in Watson-Crick type base-pairing between inosine and cytidine with a potential bifurcated hydrogen bond between inosine N1 and cytidine N3 (2.9 A) and O2 (3.3-3.A).     

Conformation of B-DNA containing O6-ethyl-G-C base pairs stabilized by minor groove binding drugs: molecular structure of d(CGC[e6G]AATTCGCG complexed with Hoechst 33258 or Hoechst 33342.

O6-ethyl-G (e6G) is an important DNA lesion, caused by the exposure of cells to alkylating agents such as N-ethyl-N-nitrosourea. A strong correlation exists between persistence of e6G lesion and subsequent carcinogenic conversion. We have determined the three-dimensional structure of a DNA molecule incorporating the e6G lesion by X-ray crystallography. The DNA dodecamer d(CGC[e6G]AATTCGCG), complexed to minor groove binding drugs Hoechst 33258 or Hoechst 33342, has been crystallized in the space group P212121, isomorphous to other related dodecamer DNA crystals. In addition, the native dodecamer d(CGCGAATTCGCG) was crystallized with Hoechst 33342. All three new structures were solved by the molecular replacement method and refined by the constrained least squares procedure to R-factors of approximately 16% at approximately 2.0 A resolution. In the structure of three Hoechst drug-dodecamer complexes in addition to the one published earlier [Teng et al. (1988) Nucleic Acids Res., 16, 2671-2690], the Hoechst molecule lies squarely at the central AATT site with the ends approaching the G4-C21 and the G16-C9 base pairs, consistent with other spectroscopic data, but not with another crystal structure reported [Pjura et al. (1987) J. Mol. Biol., 197, 257-271]. The two independent e6G-C base pairs in the DNA duplex adopt different base pairing schemes. The e6G4-C21 base pair has a configuration similar to a normal Watson-Crick base pair, except with bifurcated hydrogen bonds between e6G4 and C21, and the ethyl group is in the proximal orientation. In contrast, the e6G16-C9 base pair adopts a wobble configuration and the ethyl group is in the distal orientation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2-A resolution

The crystal structure of a daunomycin-d(CGTACG) complex has been solved by X-ray diffraction analysis and refined to a final R factor of 0.175 at 1.2-A resolution. The crystals are in a tetragonal crystal system with space group P4(1)2(1)2 and cell dimensions of a = b = 27.86 A and c = 52.72 A. The self-complementary DNA forms a six base pair right-handed double helix with two daunomycin molecules intercalated in the d(CpG) sequences at either end of the helix. Daunomycin in the complex has a conformation different from that of daunomycin alone. The daunomycin aglycon chromophore is oriented at right angles to the long dimension of the DNA base pairs, and the cyclohexene ring A rests in the minor groove of the double helix. Substituents on this ring have hydrogen-bonding interactions to the base pairs above and below the intercalation site. O9 hydroxyl group of the daunomycin forms two hydrogen bonds with N3 and N2 of an adjacent guanine base. Two bridging water molecules between the drug and DNA stabilize the complex in the minor groove. In the major groove, a hydrated sodium ion is coordinated to N7 of the terminal guanine and the O4 and O5 of daunomycin with a distorted octahedral geometry. The amino sugar lies in the minor groove without bonding to the DNA. The DNA double helix is distorted with an asymmetrical rearrangement of the backbone conformation surrounding the intercalator drug. The sugar puckers are C1,C2'-endo, G2,C1'-endo, C11,C1'-endo, and G12,C3'-exo. Only the C1 residue has a normal anti-glycosyl torsion angle (chi = -154 degrees), while the other three residues are all in the high anti range (average chi = -86 degrees). This structure allows us to identify three principal functional components of anthracycline antibiotics: the intercalator (rings B-D), the anchoring functions associated with ring A, and the amino sugar. The structure-function relationships of daunomycin binding to DNA as well as other related anticancer drugs are discussed.     

Effects of 5-fluorouracil/guanine wobble base pairs in Z-DNA: molecular and crystal structure of d(CGCGFG).

The chemotherapeutic agent 5-fluorouracil is a DNA base analogue which is known to incorporate into DNA in vivo. We have solved the structure of the oligonucleotide d(CGCGFG), where F is 5-fluorouracil (5FU). The DNA hexamer crystallizes in the Z-DNA conformation at two pH values with the 5FU forming a wobble base pair with guanine in both crystal forms. No evidence of the enol or ionized form of 5FU is found under either condition. The crystals diffracted X-rays to a resolution of 1.5 A and their structures have been refined to R-factors of 20.0% and 17.2%, respectively, for the pH = 7.0 and pH = 9.0 forms. By comparing this structure to that of d(CGCGCG) and d(CGCGTG), we were able to demonstrate that the backbone conformation of d(CGCGFG) is similar to that of the archetypal Z-DNA. The two F-G wobble base pairs in the duplex are structurally similar to the T-G base pairs both with respect to the DNA helix itself and its interactions with solvent molecules. In both cases water molecules associated with the wobble base pairs bridge between the bases and stabilize the structure. The fluorine in the 5FU base is hydrophobic and is not hydrogen bonded to any solvent molecules.     

Interaction between the Z-type DNA duplex and 1,3-propanediamine: crystal structure of d(CACGTG)2 at 1.2 A resolution

The crystal structure of a hexamer duplex d(CACGTG)(2) has been determined and refined to an R-factor of 18.3% using X-ray data up to 1.2 A resolution. The sequence crystallizes as a left-handed Z-form double helix with Watson-Crick base pairing. There is one hexamer duplex, a spermine molecule, 71 water molecules, and an unexpected diamine (Z-5, 1,3-propanediamine, C(3)H(10)N(2)) in the asymmetric unit. This is the high-resolution non-disordered structure of a Z-DNA hexamer containing two AT base pairs in the interior of a duplex with no modifications such as bromination or methylation on cytosine bases. This structure does not possess multivalent cations such as cobalt hexaammine that are known to stabilize Z-DNA. The overall duplex structure and its crystal interactions are similar to those of the pure-spermine form of the d(CGCGCG)(2) structure. The spine of hydration in the minor groove is intact except in the vicinity of the T5A8 base pair. The binding of the Z-5 molecule in the minor grove of the d(CACGTG)(2) duplex appears to have a profound effect in conferring stability to a Z-DNA conformation via electrostatic complementarity and hydrogen bonding interactions. The successive base stacking geometry in d(CACGTG)(2) is similar to the corresponding steps in d(CG)(3). These results suggest that specific polyamines such as Z-5 could serve as powerful inducers of Z-type conformation in unmodified DNA sequences with AT base pairs. This structure provides a molecular basis for stabilizing AT base pairs incorporated into an alternating d(CG) sequence.     

Molecular structure of an A-DNA decamer d(ACCGGCCGGT)

The molecular structure of the DNA decamer d(ACCGGCCGGT) has been solved and refined by single-crystal X-ray-diffraction analysis at 0.20 nm to a final R-factor of 18.0%. The decamer crystallizes as an A-DNA double helical fragment with unit-cell dimensions of a = b = 3.923 nm and c = 7.80 nm in the space group P6(1)22. The overall conformation of this A-DNA decamer is very similar to that of the fiber model for A-DNA which has a large average base-pair tilt and hence a wide and shallow minor groove. This structure is in contrast to that of several A-DNA octamers in which the molecules all have low base-pair-tilt angles (8-12 degrees) resulting in an appearance intermediate between B-DNA and A-DNA. The average helical parameters of this decamer are typical of A-DNA with 10.9 base pairs/turn of helix, an average helical twist angle of 33.1 degrees, and a base-pair-tilt angle of 18.2 degrees. However, the CpG step in this molecule has a low local-twist angle of 24.5 degrees, similar to that seen in other A-DNA oligomers, and therefore appears to be an intrinsic stacking pattern for this step. The molecules pack in the crystal using a recurring binding motif, namely, the terminal base pair of one helix abuts the surface of the shallow minor groove of another helix. In addition, the GC base pairs have large propeller-twist angles, unlike those found most other A-DNA structures.     

Molecular structure of (m5 dC-dG)3: the role of the methyl group on 5-methyl cytosine in stabilizing Z-DNA.

The hexamer (m5 dC-dG)3 has been synthesized and its three-dimensional structure determined by a single crystal X-ray diffraction analysis. The structure has been refined to a final R value of 15.6% at 1.3 A resolution. The molecule forms a left-handed Z-DNA helix which is similar to the unmethylated Z-DNA structure. The presence of the methyl group has resulted in slight changes in the twist angle between successive base pairs and modification of some of the interatomic contacts. Methylation of cytosine in the C5 position is associated with a relative destabilization of the B-DNA structure and a stabilization through hydrophobic bonding of the Z-DNA structure.     

AT base pairs are less stable than GC base pairs in Z-DNA: The crystal structure of d(m5CGTAm5CG)

Two hexanucleoside pentaphosphates, 5-methyl and 5-bromo cytosine derivatives of d(CpGpTp-ApCpG) have been synthesized, crystallized, and their three-dimensional structure solved. They both form left-handed Z-DNA and the methylated derivative has been refined to 1.2 Å resolution. These are the first crystal Z-DNA structures that contain AT base pairs. The overall form of the molecule is very similar to that of the unmethylated or the fully methylated (dC-dG)₃ hexamer although there are slight changes in base stacking. However, significant differences are found in the hydration of the helical groove. When GC base pairs are present, the helical groove is systematically filled with two water molecules per base pair hydrogen bonded to the bases. Both of these water molecules are not seen in the electron density map in the segments of the helix containing AT base pairs, probably because of solvent disorder. This could be one of the features that makes AT base pairs form Z-DNA less readily than GC base pairs.     

Binding of a macrocyclic bisacridine and ametantrone to CGTACG involves similar unusual intercalation platforms

The binding of a macrocyclic bisacridine and an antitumor intercalator ametantrone to DNA has been studied. We carried out X-ray diffraction analyses of the complexes between both intercalators and CGTACG. We have determined the crystal structure, by the multiple-wavelength anomalous diffraction (MAD) method, of bisacridine complexed with CGTA[br(5)C]G at 1.8 A resolution. The refined native crystal structure at 1.1 A resolution (space group C222, a = 29.58 A, b = 54.04 A, c = 40.22 A, and R-factor = 0.163) revealed that only one acridine of the bisacridine drug binds at the C5pG6 step of the DNA, with the other acridine plus both linkers completely disordered. Surprisingly, both terminal G.C base pairs are unraveled. The C1 nucleotide is disordered, and the G2 base is bridged to its own phosphate P2 through a hydrated Co(2+) ion. G12 is swung toward the minor groove with its base stacked over the backbone. The C7 nucleotide is flipped away from the duplex part and base paired to a 2-fold symmetry-related G6. The central four base pairs adopt the B-DNA conformation. An unusual intercalator platform is formed by bringing four complexes together (involving the 222 symmetry) such that the intercalator cavity is flanked by two sets of G x C base pairs (i.e., C5 x G8 and G6 x C7) on each side, joined together by G6 x G8 tertiary base pairing interactions. In the bisacridine-CGTACG complex, the intercalation platform is intercalated with two acridines, whereas in the ametantrone-CGTACG complex, only one ametantrone is bound. NMR titration of the bisacridine to AACGATCGTT suggests that the bisacridine prefers to bridge more than one DNA duplex by intercalating each acridine to different duplexes. The results may be relevant in understanding binding of certain intercalators to DNA structure associated with the quadruplet helix and Holliday junction.     

A novel end-to-end binding of two netropsins to the DNA decamers d(CCCCCIIIII)2, d(CCCBr5CCIIIII)2and d(CBr5CCCCIIIII)2.

Netropsin is bound to the DNA decamer d(CCCCCIIIII)2, the C-4 bromo derivative d(CCCBr5CCIIIII)2and the C-2 bromo derivative d(CBr5CCCCIIIII)2in a novel 2:1 mode. Complexes of the native decamer and the C-4 bromo derivative are isomorphous, space group P1, unit cell dimensions a = 32.56 A (32.66), b = 32.59 A (32.77), c = 37.64 A (37.71), alpha = 86.30 degrees (86.01 degrees), beta = 84.50 degrees (84.37 degrees), gamma = 68.58 degrees (68.90 degrees) with two independent molecules (A and B) in the asymmetric unit (values in parentheses are for the derivative). The C-2 bromo derivative is hexagonal P61, unit cell dimensions a = b = 32.13 A, c = 143.92, gamma = 120 degrees with one molecule in the asymmetric unit. The structures were solved by the molecular replacement method. The novelty of the structures is that there are two netropsins bound end-to-end in the minor groove of each B-DNA decamer which has nearly a complete turn. The netropsins are held by hydrogen bonding interactions to the base atoms and by sandwiching van der Waal's interactions from the sugar-phosphate backbones of the double helix similar to every other drug.DNA complex. Each netropsin molecule spans approximately 5 bp. The netropsins refined with their guanidinium heads facing each other at the center, although an orientational disorder for the netropsins cannot be excluded. The amidinium ends stretch out toward the junctions and bind to the adjacent duplexes in the columns of stacked symmetry-related complexes. Both cationic ends of netropsin are bridged by water molecules in one of the independent molecules (molecule A) of the triclinic structures and also the hexagonal structure to form pseudo-continuous drug.decamer helices.     

Crystallization and preliminary analysis of the deoxyoligonucleotide d(CGTAGATCTACG)

Two crystal forms of the self-complementary DNA 12-mer d(CGTAGATCTACG) were grown by the vapour diffusion technique. Form I is in space group C2 with a = 64.8 A, b = 35.4 A, c = 24.4 A and beta = 92.2 (1 A = 0.1 nm). The crystals are grown as monoclinic blocks or hexagonal plates. There are two strands (one duplex) in the asymmetric unit. Form II crystallizes as monoclinic blocks, space group P21 with a = 64.5 A, b = 35.1 A, c = 25.2 A and beta = 91.8 degrees. This form contains four strands (2 duplexes) in the asymmetric unit. Both forms are suitable for high resolution X-ray analysis. The diffraction patterns suggest that the DNA is in a B-type conformation and that the packing in the two forms is very similar.     

Cobalt hexammine induced tautomeric shift in Z-DNA: the structure of d(CGCGCA)*d(TGCGCG) in two crystal forms

We report here the crystal structure of the DNA hexamer duplex d(CGCGCA).d(TGCGCG) at 1.71 Å resolution. The crystals, in orthorhombic space group, were grown in the presence of cobalt hexammine, a known inducer of the left-handed Z form of DNA. The interaction of this ion with the DNA helix results in a change of the adenine base from the common amino tautomeric form to the imino tautomer. Consequently the A:T base pair is disrupted from the normal Watson–Crick base pairing to a ‘wobble’ like base pairing. This change is accommodated easily within the helix, and the helical parameters are those expected for Z-DNA. When the cobalt hexammine concentration is decreased slightly in the crystallization conditions, the duplex crystallizes in a different, hexagonal space group, with two hexamer duplexes in the asymmetric unit. One of these is situated on a crystallographic 6-fold screw axis, leading to disorder. The tautomeric shift is not observed in this space group. We show that the change in inter-helix interactions that lead to the two different space groups probably arise from the small decrease in ion concentration, and consequently disordered positions for the ion.     

Structural variability and new intermolecular interactions of Z-DNA in crystals of d(pCpGpCpGpCpG).

We have determined the single crystal x-ray structure of the synthetic DNA hexamer d(pCpGpCpGpCpG) in two different crystal forms. The hexamer pCGCGCG has the Z-DNA conformation and in both cases the asymmetric unit contains more than one Z-DNA duplex. Crystals belong to the space group C222(1) with a = 69.73, b = 52.63, and c = 26.21 A, and to the space group P2(1) with a = 49.87, b = 41.26, c = 21.91 A, and gamma = 97.12 degrees. Both crystals show new crystal packing modes. The molecules also show striking new features when compared with previously determined Z-DNA structures: 1) the bases in one duplex have a large inclination with respect to the helical axis, which alters the overall shape of the molecule. 2) Some cytosine nitrogens interact by hydrogen bonding with phosphates in neighbor molecules. Similar base-phosphate interactions had been previously detected in some B-DNA crystals. 3) Basepair stacking between the ends of neighbor molecules is variable and no helical continuity is maintained between contiguous hexamer duplexes.