The crystallographic study of left-handed Z-DNA d(CGCGCG)2 and thermine complexes crystallized at various temperatures and at various concentration of cations

In crystals of complexes of thermine and d(CGCGCG)₂ molecules grown at 4, 10, and 20 °C, the numbers of thermine molecules connected to the DNA molecule were dependent on the temperature of the crystallization. Two molecules of thermine and one Mg²⁺ ion were connected to DNA molecule when thermine and d(CGCGCG)₂ were co-crystallized at 4 and at 20 °C. When an increased concentration of magnesium and thermine molecules were co-crystallized with d(CGCGCG)₂ molecules at 10 °C, three Mg²⁺ ions and only one thermine molecule were bound with a d(CGCGCG)₂ molecule. The number of polyamines and of Mg²⁺ ions connected to DNA was dependent on the atomic values of the polyamine and of the metal ion. The binding of more Mg²⁺ ions occurred when the atomic value of Mg²⁺ exceeded that of the corresponding mono- or polyamine, and when the Mg²⁺ ion concentration was elevated. Furthermore, this study is the first documentation of a naturally occurring polyamine bound to the minor groove of DNA in a crystal structure.     

The rare crystallographic structure of d(CGCGCG)(2): the natural spermidine molecule bound to the minor groove of left-handed Z-DNA d(CGCGCG)(2) at 10 degrees C

Several crystal structure analyses of complexes of synthetic polyamine compounds, including N(1)-(2-(2-aminoethylamino))ethyl)ethane-1,2-diamine PA(222) and N(1)-(2-(2-(2-aminoethylamino)ethylamino)ethyl)ethane-1,2-diamine PA(2222), and left-handed Z-DNA d(CGCGCG)(2) have been reported. However, until now, there have been no examples of naturally occurring polyamines bound to the minor groove of the left-handed Z-DNA of d(CGCGCG)(2) molecule. We have found that spermidine, a natural polyamine, is connected to the minor groove of left-handed Z-DNA of d(CGCGCG)(2) molecule in a crystalline complex grown at 10 degrees C. The electron density of the DNA molecule was clear enough to determine that the spermidine was connected in the minor groove of two symmetry related molecules of left-handed Z-DNA d(CGCGCG)(2). This is the first example that a spermidine molecule can form a bridge conformation between two symmetry related molecules of left-handed Z-DNA d(CGCGCG)(2) in the minor groove.     

Flexibility and rigidity of left-handed Z-DNA

The local variation of torsional angles and helical parameters in Z-DNA was analyzed. The sugar phosphate backbone is fairly rigid but the angles at GpC linkage are more changeable than those at CpG linkage in order to form a variety of structures. The water channel at minor groove is important to stabilize and retain the novel Z-DNA helix.     

The molecular structure of the left-handed Z-DNA double helix at 1.0-A atomic resolution. Geometry, conformation, and ionic interactions of d(CGCGCG)

The structure of d(CGCGCG) crystallized in the presence of magnesium and sodium ions alone is compared to that of the spermine form of the molecule. The very high resolution nature of these structure determinations allows the first true examination of an oligonucleotide structure in fine detail. The values of bond distances and angles are compared to those derived from small molecule crystal structures. In addition, the interactions of cations and polyamines with the Z-DNA helix are analyzed. In particular, multiple cationic charges appear to offer enhanced stabilization for the Z-DNA conformation. The location of spermine molecules along the edge of the deep groove and also spanning the entrance to the groove emphasizes the importance of polyamines for stabilizing this left-handed structure. On averaging, we obtained very similar structural parameters for the two different structures with standard deviations generally smaller than the deviations of the crystallographic model from ideal values. This indicates a high degree of accuracy of the two structures, which have been refined using different data and different refinement methods. The derived bond lengths and angles may thus be more representative of this polymeric DNA structure than those derived from mono- and dinucleotide structures at a similar accuracy.     

Structure of the pure-spermine form of Z-DNA (magnesium free) at 1-A resolution.

We describe the three-dimensional X-ray structure of a complex of spermine bound to a Z-DNA duplex, [d(CGCGCG)]2, in the absence of any inorganic polyvalent cations. We have crystallized the DNA hexamer d(CGCGCG) in the exclusion of magnesium and other polyvalent ions and solved its structure at 1.0-A resolution. In the crystal of this pure-spermine form of Z-DNA, the relative orientation, position, and interactions of the DNA differ from the arrangement uniformly observed in over a dozen previously reported Z-DNA hexamers. Moreover, the conformation of the Z-DNA hexamer in this structure varies somewhat from those found in earlier structures. The DNA is compressed along the helical axis, the base pairs are shifted into the major groove, and the minor groove is more narrow. The packing of spermine-DNA complexes in crystals suggests that the molecular basis for the tendency of spermine to stabilize compact DNA structures derives from the capacity of spermine to interact simultaneously with several duplexes. This capacity is maximized by both the polymorphic nature and the length of the spermine cation. The length and flexibility of spermine and the dispersion of charge-charge, hydrogen-bonding, and hydrophobic bonding potential throughout the molecule maximize the ability of spermine to interact simultaneously with different DNA molecules.     

Z-DNA crystallography

We review the effect of sequence on the structure of left-handed Z-DNA in single crystals. The various substituent groups that define a nucleotide base as guanine, cytosine, thymine, or adenine affect both the DNA conformation and the organization of solvent around the duplex. These are discussed in terms of their effect on the ability of sequences to adopt the unusual Z-DNA structure. In addition, the experimental and theoretical methods used to treat DNA hydration are discussed as they relate to the stability of Z-DNA. Finally, we argue that Z-DNA, as defined by the crystal conformation, is sufficient in itself to account for the physical properties of left-handed conformations observed in polymers and in genomic sequences. © 1997 John Wiley & Sons, Inc. Biopoly 44: 65–90, 1997     

Water structure around a left-handed Z-DNA fragment analyzed by cryo neutron crystallography

Even in high-quality X-ray crystal structures of oligonucleotides determined at a resolution of 1 Å or higher, the orientations of first-shell water molecules remain unclear. We used cryo neutron crystallography to gain insight into the H-bonding patterns of water molecules around the left-handed Z-DNA duplex [d(CGCGCG)]₂. The neutron density visualized at 1.5 Å resolution for the first time allows us to pinpoint the orientations of most of the water molecules directly contacting the DNA and of many second-shell waters. In particular, H-bond acceptor and donor patterns for water participating in prominent hydration motifs inside the minor groove, on the convex surface or bridging nucleobase and phosphate oxygen atoms are finally revealed. Several water molecules display entirely unexpected orientations. For example, a water molecule located at H-bonding distance from O6 keto oxygen atoms of two adjacent guanines directs both its deuterium atoms away from the keto groups. Exocyclic amino groups of guanine (N2) and cytosine (N4) unexpectedly stabilize waters H-bonded to O2 keto oxygens from adjacent cytosines and O6 keto oxygens from adjacent guanines, respectively. Our structure offers the most detailed view to date of DNA solvation in the solid-state undistorted by metal ions or polyamines.     

Stabilization of Z-DNA by demethylation of thymine bases: 1.3-A single-crystal structure of d(m5CGUAm5CG)

Methylation of cytosine bases at the C5 position has been known to stabilize Z-DNA. We had previously predicted from calculations of solvent-accessible surfaces that the methyl group at the same position of thymine has a destabilizing effect on Z-DNA. In the current studies, the sequence d(m5CGUAm5CG) has been crystallized and its structure solved as Z-DNA to 1.3-A resolution. A well-defined octahedral hexaaquomagnesium complex was observed to bridge the O4 oxygens of the adjacent uridine bases at the major groove surface, and four well-structured water molecules were found in the minor groove crevice at the d(UA) dinucleotide. These solvent interactions were not observed in the previously published Z-DNA structure of the analogous d(m5CGTAm5CG) sequence. A comparison of the thymine and uridine structures supports our prediction that demethylation of thymine bases helps to stabilize Z-DNA. A comparison of this d(UA)-containing Z-DNA structure with the analogous d(TA) structure shows that access of the O4 position is hindered by the C5 methyl of thymine due to steric and hydrophobic inhibition. In the absence of the methyl group, a magnesium-water complex binds to and slightly affects the structure of the Z-DNA major groove surface. This perturbation of the solvent structure at the major groove surface is translated into a much larger 1.41-A widening of the minor groove crevice, thereby allowing the specific binding of two water molecules at well-defined sites of each internal d(UA) base pair. Possible mechanisms by which modifications at the major groove surface of Z-DNA can affect the solvent properties of the minor groove crevice are discussed.     

Recognition of Z-RNA and Z-DNA determinants by polyamines in solution: experimental and theoretical studies

Protonated polyamines are among the most efficient cations that induce the left-handed Z-form in certain polynucleotides. It is not known, however, whether these cations bind to specific sites on Z-sequences in solution. We have studied potential polyamine binding sites by measuring the effects of polyamines on the binding of purified immunoglobulins (IgGs) to different regions of the Z-helix and by molecular mechanics modeling. The specific binding of anti-Z-DNA and anti-Z-RNA IgGs to Z-helices was studied as a function of spermidine or spermine concentration. The effect of polyamines on the antibody-nucleic acid interaction was different for IgGs with different specificities for various determinants on the Z-helix. Polyamines inhibit the binding of certain anti-Z IgGs directed against specific sites probably at or near the interface between the major convex surface and the phosphate backbone, most likely by competing with the antibody binding site(s). In contrast, polyamines have no effect on other anti-Z IgGs directed against sites determined by the phosphate backbone. Furthermore, these cations can enhance the binding of anti-Z IgG directed against bulky groups at the C-5 position on the major convex surface of the helix; the enhancement may be related to charge neutralization. Under these conditions, no direct binding of antibodies with polyamines was observed. These data suggest the existence of a specific binding site(s) for polyamines on both Z-DNA and Z-RNA in solution. These binding sites have some similarity to those observed in oligonucleotide crystals by Quigley (in "Molecular Structure and Biological Activity," J.F. Griffin and W.L. Duax, eds., Elsevier, Amsterdam (1982), pp. 317-331). The experimental evidence for specific spermine binding sites on the helical surface was supported by molecular mechanics modeling of the interaction of spermine with the major groove of (dG-dC)5.(dG-dC)5 in both the Z- and B-forms. The crystal coordinates of spermine-containing oligonucleotides in both the B- and Z-forms were used as the starting points for modeling studies. The potential energy of spermine bound to the major convex surface of the Z-form was much less favorable than that of spermine bound to the major groove of the B-form. In the presence of sodium ions, however, the Z-form-spermine complexes were favored over the B-form. Thus, both theoretical and experimental studies indicate that polyamines can specifically recognize Z-helical determinants in solution as well as in crystals.     

The Computational Studies on the Conformation-Stabilizing Factors of the Left-Handed Z-DNA

Abstract

The conformation-stabilizing factors of the left-handed Z-DNA was estimated by the molecular dynamics calculations using the various metal cations and the polyamines. We concluded the conformation-stabilizing effect of metal cation for Z-DNA was more significant than that of the polyamine by theoretically.     

Crystal structure of the self-complementary 5''-purine start decamer d(GCGCGCGCGC) in the Z-DNA conformation. I.

Alternating self-complementary oligonucleotides starting with a 5'-pyrimidine usually form left-handed Z-DNA; however, with a 5'-purine start sequence they form the right-handed A-DNA. Here we report the crystal structure of the decamer d(GCGCGCGCGC) with a 5'-purine start in the Z-DNA form. The decamer crystallizes in the hexagonal space group P6(5)22, unit cell dimensions a = b = 18.08 and c = 43.10 A, with one of the following four dinucleotide diphosphates in the asymmetric unit: d(pGpC)/d(GpCp)/d(pCpG)/d(CpGp). The molecular replacement method, starting with d(pGpC) of the isomorphous Z-DNA hexamer d(araC-dG)3 without the 2'-OH group of arabinose, was used in the structure analysis. The method gave the solution only after the sugar-phosphate conformation of the GpC step was manipulated. The refinement converged to a final R value of 18.6% for 340 unique reflections in the resolution range 8.0-1.9 A. A result of the sequence alternation is the alternation in the nucleotide conformation; guanosine is C3'-endo, syn, and cytidine is C2'-endo, anti. The CpG step phosphodiester conformation is the same as ZI or ZII, whereas that of the GpC step phosphodiester is "intermediate" in the sense that zeta (O3'-P bond) is the same as ZII but alpha (P-O5' bond) is the same as ZI. The duplexes generated from the dinucleotide asymmetric unit are stacked one on top of the other in the crystal to form an infinite pseudocontinuous helix. This renders it a quasi-polymerlike structure that has assumed the Z-DNA conformation further strengthened by the long inner Z-forming stretch d(CG)4. An interesting feature of the structure is the presence of water strings in both the major and the minor grooves. In the minor groove the cytosine carbonyl oxygen atoms of the GpC and CpG steps are cross-bridged by water molecules that are not themselves hydrogen bonded but are enclosed by the water rings in the mouth of the minor groove. In the major groove three independent water molecules form a zigzagging continuous water string that runs throughout the duplex.     

Reduced 4,5',8-trimethylpsoralen cross-linking of left-handed Z-DNA stabilized by DNA supercoiling

Z-DNA-forming sequences, (GT)21, (GT)12ATGT, and (CG)6TA(CG)6, were cloned into plasmids. These sequences formed left-handed Z-DNA conformations under torsional tension from negative supercoiling of DNA. 4,5',8-Trimethylpsoralen, on absorption of 360-nm light, forms monoadducts and interstrand cross-links in DNA that exists in the B-helical conformation. Trimethylpsoralen cross-links were introduced into the potential Z-DNA-forming sequences in relaxed DNA when these sequences existed as B-form DNA. In supercoiled DNA when these sequences existed in the Z conformation, the rate of cross-linking was greatly reduced, and trimethylpsoralen did not form monoadducts appreciably to Z-DNA. As an internal control in these experiments, the rates of cross-linking of the Z-DNA-forming sequences were measured relative to that of an adjacent, cloned sequence that could not adopt a Z conformation. The initial relative rates of cross-linking to Z-DNA-forming sequences were dependent on the superhelical density of the DNA, and the rates were ultimately reduced by factors of 10-15 for Z-DNA in highly supercoiled plasmids. This differential rate of cross-linking provides a novel assay for Z-DNA. Initial application of this assay in vivo suggests that a substantial fraction of (CG)6TA(CG)6, which existed as Z-DNA in plasmid molecules purified from cells, existed in the B conformation in vivo.     

Structure of d(CACGTG), a Z-DNA hexamer containing AT base pairs.

The left-handed Z-DNA conformation has been observed in crystals made from the self-complementary DNA hexamer d(CACGTG). This is the first time that a non disordered Z form is found in the crystal structure of an alternating sequence containing AT base pairs without methylated or brominated cytosines. The structure has been determined and refined to an agreement factor R = 22.9% using 746 reflections in the resolution in the resolution shell 7 to 2.5 A. The overall shape of the molecule is very similar to the Z-structure of the related hexamer d(CG)3 confirming the rigidity of the Z form. No solvent molecules were detected in the minor groove of the helix near the A bases. The disruption of the spine of hydration in the AT step appears to be a general fact in the Z form in contrast with the B form. The biological relevance of the structure in relation to the CA genome repeats is discussed.     

Structural specificity of polyamines in left-handed Z-DNA formation. Immunological and spectroscopic studies

Natural polyamines putrescine, spermidine and spermine are ubiquitous cellular components. Recent studies showed that these compounds are capable of provoking a conformational transition in poly(dG-m5dC).poly(dG-m5dC) from its usual right-handed B-DNA form to a left-handed Z-DNA form at physiologically relevant cationic concentrations. We studied the efficacy of spermidine, six homologs of spermidine (H2N(CH2)nNH(CH2)3NH2, where n = 2 to 8 (n = 4 for spermidine)) and diethylene triamine to provoke the B-DNA to Z-DNA transition of poly(dG-m5dC).poly(dG-m5dC) using a monoclonal anti-Z-DNA antibody and spectroscopic techniques. The concentration of spermidine at the midpoint of B-DNA to Z-DNA transition was 30 +/- 1 microM. Chemical structural effects were significant when the spermidine homologs were used to induce the transition. The midpoint concentration increased as the number of -CH2 groups varied in relation to that of spermidine. We interpret these structural effects on the basis of molecular models of the interaction of polyamines with polynucleotides.     

Non-contiguous regions of Z-DNA in a DNA dodecamer.

The conformation of the self-complimentary DNA dodecamer d(br5CGbr5CGAATTbr5CGbr5CG) has been investigated in a variety of salt and solvent conditions by one and two-dimensional 1H NMR. In low salt aqueous solutions, the molecule forms a regular B-DNA structure similar to the unmodified dodecamer. However, in aqueous solution containing high salt concentration and methanol, the dodecamer adopts a structure in which the br5CGbr5CG ends of the molecule are in a Z-DNA like conformation and the AATT region is neither standard B-DNA nor Z-DNA. The implications of these results for the structure of junctions between B and Z-DNA and the sequence specificity of Z-DNA are discussed.     

Polyamine-induced B-DNA to Z-DNA conformational transition of a plasmid DNA with (dG-dC)n insert.

We investigated the ability of natural polyamines putrescine, spermidine, and spermine to provoke a left-handed Z-DNA conformation in a recombinant plasmid (pDHg16) with a 23-base pair insert of (dG-dC)n.(dG-dC)n sequences. Using a monoclonal anti-Z-DNA antibody (Z22) and an enzyme-linked immunosorbent assay protocol, we found that spermidine and spermine were capable of converting pDHg16 to the Z-DNA form. The concentrations of spermidine and spermine at the midpoint of the B-DNA to Z-DNA transition were 280 and 5 microM, respectively, in buffer containing 50 mM NaCl, 1 mM sodium cacodylate, and 0.15 mM EDTA, pH 7.4. A plot of ln[Na+] versus ln [spermine4+], where [Na+] is the bulk NaCl concentration and [spermine4+] is the spermine concentration at the midpoint of the B-DNA to Z-DNA transition, gave a straight line with a slope of 1.2. Structural specificity was clearly evident in the efficacy of three spermidine homologs to induce the Z-DNA conformation in pDHg16. Putrescine and acetylspermidines had no effect on the conformation of the plasmid DNA up to a 3 mM concentration. Control experiments with the parental plasmid (pDPL6) showed no binding of the plasmid DNA with Z22. These results indicate that spermidine and spermine are capable of provoking the left-handed Z-DNA conformation in small blocks of (dG-dC)n sequences embedded in a right-handed B-DNA matrix. Since blocks of (dG-dC)n sequences are found in certain native DNAs, conformational alterations of these regions to the Z-DNA form in the presence of polyamines may have important gene regulatory effects.     

Effects of base substituents on the hydration of B- and Z-DNA: correlations to the B- to Z-DNA transition.

We present a study of how substituent groups of naturally occurring and modified nucleotide bases affect the degree of hydration of right-handed B-DNA and left-handed Z-DNA. A comparison of poly(dG-dC) and poly(dG-dm5C) titrations with the lipotropic salts of the Hofmeister series infers that the methyl stabilization of cytosines as Z-DNA is primarily a hydrophobic effect. The hydration free energies of various alternating pyrimidine-purine sequences in the two DNA conformations were calculated as solvent free energies from solvent accessible surfaces. Our analysis focused on the N2 amino group of purine bases that sits in the minor groove of the double helix. Removing this amino group from guanine to form inosine (I) destabilizes Z-DNA, while adding this group to adenines to form 2-aminoadenine (A') stabilizes Z-DNA. These predictions were tested by comparing the salt concentrations required to crystallize hexanucleotide sequences that incorporate d(CG), d(CI), d(TA) and d(TA') base pairs as Z-DNA. Combining the current results with our previous analysis of major groove substituents, we derived a thermodynamic cycle that relates the systematic addition, deletion, or substitution of each base substituent to the B- to Z-DNA transition free energy.     

Chemical footprinting of the interaction between left-handed Z-DNA and anti-Z-DNA antibodies by diethylpyrocarbonate carbethoxylation

Diethylpyrocarbonate (DEPC) carbethoxylates Z-DNA to an increased extent because the reactive N-7 atoms of purine residues appear structurally more accessible on Z-DNA as opposed to B-DNA. This chemical probe was used in DEPC footprinting experiments, which confirm the specificity of binding of anti-Z-DNA monoclonal antibodies and which probe regions of close contact in this DNA-protein complex. Antibody binding to segments of Z-DNA existing in supercoiled plasmids resulted in specific protection from DEPC hyper-reactivity within the Z-DNA segment and induction of hyper-reactivity in purines lying adjacent to the Z-segment. Two different monoclonal immunoglobulin preparations, Z22 and Z44, are shown to generate specific and distinct footprint patterns when bound to the Z-helix. Binding of these antibodies was also found to affect DNA conformation within the Z-DNA segment by influencing the equilibrium between the B- and Z-helical conformations.