The interaction between Z-DNA and the Zab domain of double-stranded RNA adenosine deaminase characterized using fusion nucleases.

Zab is a structurally defined protein domain that binds specifically to DNA in the Z conformation. It consists of amino acids 133-368 from the N terminus of human double-stranded RNA adenosine deaminase, which is implicated in RNA editing. Zab contains two motifs with related sequence, Zalpha and Zbeta. Zalpha alone is capable of binding Z-DNA with high affinity, whereas Zbeta alone has little DNA binding activity. Instead, Zbeta modulates Zalpha binding, resulting in increased sequence specificity for alternating (dCdG)n as compared with (dCdA/dTdG)n. This relative specificity has previously been demonstrated with short oligonucleotides. Here we demonstrate that Zab can also bind tightly to (dCdG)n stabilized in the Z form in supercoiled plasmids. Binding was assayed by monitoring cleavage of the plasmids using fusion nucleases, in which Z-DNA-binding peptides from the N terminus of double-stranded RNA adenosine deaminase are linked to the nuclease domain of FokI. A fusion nuclease containing Zalpha shows less sequence specificity, as well as less conformation specificity, than one containing Zab. Further, a construct in which Zbeta has been replaced in Zab with Zalpha, cleaves Z-DNA regions in supercoiled plasmids more efficiently than the wild type but with little sequence specificity. We conclude that in the Zab domain, both Zalpha and Zbeta contact DNA. Zalpha contributes contacts that produce conformation specificity but not sequence specificity. In contrast, Zbeta contributes weakly to binding affinity but discriminates between sequences of Z-DNAs.     

The Zβ domain of human DAI binds to Z-DNA via a novel B-Z transition pathway

The human DNA-dependent activator of IFN-regulatory factor (DAI) protein, which activates the innate immune response in response to DNA, contains two tandem Z-DNA binding domains (Zα and Zβ) at the NH(2) terminus. The hZβ(DAI) structure is similar to other Z-DNA binding proteins, although it demonstrates an unusual Z-DNA recognition. We performed NMR experiments on complexes of hZβ(DAI) with DNA duplex, d(CGCGCG)(2), at a variety of protein-to-DNA molar ratios. The results suggest that hZβ(DAI) binds to Z-DNA via an active-di B-Z transition mechanism, where two hZβ(DAI) proteins bind to B-DNA to form the hZβ(DAI)-B-DNA complex; the B-DNA is subsequently converted to left-handed Z-DNA. This novel mechanism of DNA binding and B-Z conversion is distinct from Z-DNA binding of the human ADAR1 protein.     

Anti-Z-DNA antibody binding can stabilize Z-DNA in relaxed and linear plasmids under physiological conditions.

It is shown that anti-Z-DNA antibody binding can stabilize sequences of d(CG/GC)n and d(CA/GT)n in the Z-DNA conformation in a plasmid in the complete absence of supercoiling. This effect is quantitated by using antibody preparations of different affinities and varying concentrations. The d(CG/GC)n sequence can be stabilized under physiological conditions. This is the first demonstration that a region of Z-DNA can be stabilized by protein binding in a completely relaxed plasmid under physiological conditions. The antibody-Z-DNA complex in the relaxed plasmid is shown to be an equilibrium state and not a long-lived kinetic intermediate since specific binding of the antibody to linearized plasmids containing Z-forming sequences is observed.     

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.     

Isolation and characterization of Z-DNA binding proteins from wheat germ

The preparation of a heterogeneous non-histone protein extract from wheat germ utilizing Br-poly(dG-dC).poly(dG-dC) (Z-DNA) affinity chromatography is described. The binding characteristics of antibodies against Z-DNA are used as a model system to define important criteria that the DNA binding behavior of a Z-DNA binding protein should display. We show that the wheat germ extract contains DNA binding proteins specific for left-handed Z-DNA by these criteria. The affinity of the proteins measured by competition experiments was approximately 10(5) greater for Br-poly(dG-dC).poly(dG-dC) (Z-DNA) than for poly(dG-dC).poly(dG-dC) (B-DNA). The affinity of the proteins for plasmid DNA increases with increasing negative superhelicity which is known to stabilize Z-DNA. The proteins are shown to compete with Z-DNA antibodies for binding to supercoiled plasmids. Finally, the affinity for two plasmids at a given superhelical density is greater for the plasmid containing an insert known to form Z-DNA than for a plasmid without the insert. The proteins exhibit a 2-3-fold greater affinity for stretches of (dC-dA)n.(dT-dG)n over stretches of (dG-dC)n.(dG-dC)n when both sequences are induced to form Z-DNA by supercoiling.     

In vivo assessment of the Z-DNA-forming potential of d(CA.GT)n and d(CG.GC)n sequences cloned into SV40 minichromosomes

Alternating repeated d(CA.GT)n and d(CG.GC)n sequences constitute a significant proportion of the simple repeating elements found in eukaryotic genomic DNA. These sequences are known to form left-handed Z-DNA in vitro. In this paper, we have addressed the question of the in vivo determination of the Z-DNA-forming potential of such sequences in eukaryotic chromatin. For this purpose, we have investigated the ability of a d(CA.GT)30 sequence and a d(CG.GC)5 sequence to form left-handed Z-DNA when cloned into simian virus 40 (SV40) minichromosomes at two different positions: the TaqI site, which occurs in the intron of the T-antigen gene, and the HpaII site, which is located in the late promoter region within the SV40 control region. Formation of Z-DNA at the inserted repeated sequences was analyzed through the change in DNA linkage associated with the B to Z transition. Our results indicate that regardless of: (1) the site of insertion (either TaqI or HpaII), (2) the precise moment of the viral lytic cycle (from 12 h to 48 h postinfection) and (3) the condition of incorporation of the SV40 recombinants to the host cells (either as minichromosomes or as naked DNA, relaxed or negatively supercoiled), neither the d(CA.GT)30 nor the d(CG.GC)5 sequence are stable in the left-handed Z-DNA conformation in the SV40 minichromosome. The biological relevance of these results is 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.     

Stabilization of Z-DNA by polyarginine near physiological ionic strength.

The identification of left handed or Z-DNA in solutions of poly d(GC) in high salt suggests that left handed DNA may exist in biological systems if stabilized at lower ionic strength. In the present study we show that binding of polyarginine to the Z form of poly d(GC) results in a protein-Z-DNA complex stable near physiological ionic strength. The percentage of Z-DNA in the low salt polyarginine-poly d(GC) complex was measured from the DNA circular dichroism spectrum. The ratio of Z to B-DNA is a linear function of polyarginine concentration and is sensitive to proteolytic digestion by trypsin. These results suggest that arginine-rich proteins may stabilize Z-DNA in vivo.     

A peptide with alternating lysines can act as a highly specific Z-DNA binding domain

Many nucleic acid binding proteins use short peptide sequences to provide specificity in recognizing their targets, which may be either a specific sequence or a conformation. Peptides containing alternating lysine have been shown to bind to poly(dG–d5meC) in the Z conformation, and stabilize the higher energy form [H. Takeuchi, N. Hanamura, H. Hayasaka and I. Harada (1991) FEBS Lett., 279, 253–255 and H. Takeuchi, N. Hanamura and I. Harada (1994) J. Mol. Biol., 236, 610–617.]. Here we report the construction of a Z-DNA specific binding protein, with the peptide KGKGKGK as a functional domain and a leucine zipper as a dimerization domain. The resultant protein, KGZIP, induces the Z conformation in poly(dG–d5meC) and binds to Z-DNA stabilized by bromination with high affinity and specificity. The binding of KGZIP is sufficient to convert poly(dG–d5meC) from the B to the Z form, as shown by circular dichroism. The sequence KGKGKGK is found in many proteins, although no functional role has been established. KGZIP also has potential for engineering other Z-DNA specific proteins for future studies of Z-DNA in vitro and in vivo.     

Supercoiled induced transition to the Z-DNA conformation affects the ability of a d(CG/GC)12 sequence to be organized into nucleosome-cores.

Nucleosome-cores were reconstituted by the salt-dialysis method onto closed circular pDHg16 DNA which contains a d(CG/GC)12 sequence. Alternating d(CG/GC)n sequences form left-handed Z-DNA readily when contained in negatively supercoiled DNA. We have investigated the ability of the d(CG/GC)12 sequence to be organized into nucleosome-cores when stabilized as Z-DNA through negative supercoiling. We have found that nucleosome assembly at the d(CG/GC)12 insert is prevented when the sequence is stable in the Z-conformation but it is not affected at all when the sequence adopts the right-handed B-form.     

Sequence discrimination of the Zα domain of human ADAR1 during B-Z transition of DNA duplexes

The Zα domain of human ADAR1 (Zα_ADAR1) preferentially binds Z-DNA rather than B-DNA with high binding affinity. Zα_ADAR1 binds to the Z-conformation of both non-CG-repeat DNA duplexes and a d(CGCGCG)₂ duplex similarly. We performed NMR experiments on complexes between the Zα_ADAR1 and non-CG-repeat DNA duplexes, d(CACGTG)₂ or d(CGTACG)₂, with a variety of protein-DNA molar ratios. Comparison of these results with those from the analysis of d(CGCGCG)₂ in the previous study suggests that Zα_ADAR1 exhibits the sequence preference of d(CGCGCG)₂ ? d(CACGTG)₂ > d(CGTACG)₂ through multiple sequence discrimination steps during the B-Z transition.     

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.     

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.     

Effects of the O2' hydroxyl group on Z-DNA conformation: structure of Z-RNA and (araC)-[Z-DNA

The left-handed Z structures of two hexamers [d(CG)r(CG)d(CG) and d(CG)(araC)d(GCG)] containing ribose and arabinose residues have been solved by X-ray diffraction analysis at 1.5-A resolution. Their conformations closely resemble that of the canonical Z-DNA. The O2' hydroxyl groups of both rC and araC residues form intramolecular hydrogen bonds with N2 of the 5' guanine residue and replace the bridging water molecules in the deep groove of Z-DNA, which stabilize the guanine in the syn conformation. The araC residue can be incorporated into the Z structure readily and facilitates B to Z transition, as supported by UV absorption spectroscopic studies. In contrast, in Z-RNA the ribose of the cytidine residue is twisted in order to form the respective hydrogen bond. The potential biological roles of the modified Z-DNA containing anticancer nucleoside araC and of Z-RNA are discussed.     

Dual conformational recognition by Z-DNA binding protein is important for the B–Z transition process

Left-handed Z-DNA is radically different from the most common right-handed B-DNA and can be stabilized by interactions with the Zα domain, which is found in a group of proteins, such as human ADAR1 and viral E3L proteins. It is well-known that most Zα domains bind to Z-DNA in a conformation-specific manner and induce rapid B–Z transition in physiological conditions. Although many structural and biochemical studies have identified the detailed interactions between the Zα domain and Z-DNA, little is known about the molecular basis of the B–Z transition process. In this study, we successfully converted the B–Z transition-defective Zα domain, vvZα_E3L, into a B–Z converter by improving B-DNA binding ability, suggesting that B-DNA binding is involved in the B–Z transition. In addition, we engineered the canonical B-DNA binding protein GH5 into a Zα-like protein having both Z-DNA binding and B–Z transition activities by introducing Z-DNA interacting residues. Crystal structures of these mutants of vvZα_E3L and GH5 complexed with Z-DNA confirmed the significance of conserved Z-DNA binding interactions. Altogether, our results provide molecular insight into how Zα domains obtain unusual conformational specificity and induce the B–Z transition.     

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.