Excision of cytosine hydrates from Z-DNA.

Ultraviolet irradiation of DNA produces cytosine hydrate, released as a free base by E. coli endonuclease III. Cytosine hydrate excision was investigated by assaying photoproduct release from cytosine-radiolabeled, irradiated poly(dG-dC):poly(dG-dC). Conformational shifts between B-DNA and Z-DNA were affected by heating the polymer in either nickel chloride or cobaltous chloride, and were determined by circular dichroism. Rates of enzymic cytosine hydrate release did not differ between the different substrate conformations. Irradiation of left-handed poly(dG-dC):poly(dG-dC) resulted in cytosine hydrate formation. Therefore, neither formation nor enzymic excision of ultraviolet-induced cytosine hydrates are substantially affected by these DNA conformational states.     

Left-handed Z-DNA

Since the Watson-Crick proposal of right-handed B-DNA, numerous studies have been devoted to the conformation of DNA. Both natural DNAs of heterogeneous sequences and synthetic DNAs are capable of adopting more than one conformation. The specific conformation a DNA adopts appears to depend mainly on its base sequence and its environmental conditions. For a given DNA, changes in environmental conditions can induce conformational transitions which occur according to cooperative or non-cooperative processes (for general reviews see Ref. 1a, b). Despite many results, molecular biologists did not put much emphasis on the polymorphism of DNA. The discovery of the intraconversion in helical sense between the right-handed B and left-handed Z conformers of DNA has brought a new interest in the polymorphism of DNA. It is now proposed that this polymorphism has important functions in biological reactions. A recent review, ‘The Chemistry and Biology of Left-handed Z-DNA’, by Rich et al. [2] has just been published. We here report some of the results published in 1984 on Z-DNA.     

Left-handed Z-DNA

Since the Watson-Crick proposal of right-handed B-DNA, numerous studies have been devoted to the conformation of DNA. Both natural DNAs of heterogeneous sequences and synthetic DNAs are capable of adopting more than one conformation. The specific conformation a DNA adopts appears to depend mainly on its base sequence and its environmental conditions. For a given DNA, changes in environmental conditions can induce conformational transitions which occur according to cooperative or non-cooperative processes (for general reviews see Ref. 1a, b). Despite many results, molecular biologists did not put much emphasis on the polymorphism of DNA. The discovery of the intraconversion in helical sense between the right-handed B and left-handed Z conformers of DNA has brought a new interest in the polymorphism of DNA. It is now proposed that this polymorphism has important functions in biological reactions. A recent review, 'The Chemistry and Biology of Left-handed Z-DNA', by Rich et al. has just been published. We here report some of the results published in 1984 on Z-DNA.     

Zinc Z-DNA

Circular dichroism spectra of poly(dG-dC) in the presence of some zinc complexes exhibit the characteristic inversion associated with the formation of a left handed helix. The transition of B to Z DNA is cooperative and slow. The concentration of zinc complex at the mid point of the transition is strongly dependent upon the nature of the ligand bound to zinc. The most efficient species is one with a tetradentate amine for which the mid point is observed at a zinc:nucleotide ratio of 1:24. 31P spectra of one of these complexes confirm the presence of a left handed helix.     

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     

Z-DNA功能研究的回顾和近况

自从Rich等在1979年首次发现左手螺旋的Z-DNA以后,人们对Z-DNA的研究不断深入,并推测它在基因转录、基因调控以及基因重组等方面有着重要的作用。最新研究表明Z-DNA与病毒的发病机制有关。这个研究结果可能蕴藏着关于这种另类DNA如何行使功能的答案,并可能由此成功研制能有效抗天花病毒的化合物。     

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.     

Cytosine methylation enhances Z-DNA formation in vivo.

The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays. Both the M.EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo. A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated. Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M.HhaI) in cells also carrying a dC-dG-containing plasmid. Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M.HhaI than in the absence of M.HhaI. These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E. coli. The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.     

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.     

A Z-DNA binding protein isolated from D. radiodurans

A DNA binding protein isolated from D. radiodurans changes CD-spectrum of Z-form poly(dG-dC) X poly(dG-dC). We have found that a positive band at 268 nm is converted close to that of B-form in the presence of the protein. Concomitantly, a negative band at 295 nm shown by Z-form poly(dG-dC) X poly (dG-dC) was weakened by the protein but not by albumin. Such changes in the CD-spectra were not induced by the protein and by albumin when they were mixed with Z- or B-form poly(dG-me5dC) X poly(dG-me5dC) or with B-form poly(dG-dC) X poly(dG-dC). The protein formed a complex preferentially with Z-form poly(dG-dC) X poly(dG-dC).     

H-DNA and Z-DNA in the mouse c-Ki-ras promoter.

The mouse c-Ki-ras protooncogene promoter contains a homopurine-homopyrimidine domain that exhibits S1 nuclease sensitivity in vitro. We have studied the structure of this DNA region in a supercoiled state using a number of chemical probes for non-B DNA conformations including diethyl pyrocarbonate, osmium tetroxide, chloroacetaldehyde, and dimethyl sulfate. The results demonstrate that two types of unusual DNA structures formed under different environmental conditions. A 27-bp homopurine-homopyrimidine mirror repeat adopts a triple-helical H-DNA conformation under mildly acidic conditions. This H-DNA seems to account for the S1 hypersensitivity of the promoter in vitro, since the observed pattern of S1 hypersensitivity at a single base level fits well with the H-DNA formation. Under conditions of neutral pH we have detected Z-DNA created by a (CG)5-stretch, located adjacent to the homopurine-homopyrimidine mirror repeat. The ability of the promoter DNA segment to form non-B structures has implications for models of gene regulation.     

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.     

Reactivity of B and Z-DNA towards N-acetoxy-2-acetylaminofluorene.

Poly d(G-C) d(G-C) in B-form, on one hand, and poly d(G-br5C). poly d(G-br5C) and poly d(G-m5C) . poly d(G-m5C) in Z-form, on another hand, were treated with N-AcO-[3H]AAF and the kinetics of these reactions were followed by radioactivity. Covalent binding of carcinogen to the polymers was evaluated after separation of the reacted polymers from non-reacted carcinogen by thin-layer chromatography. We found that B-form polymer reacts twice faster than the Z-form polymers. Proportions of main adducts in the three polymers are almost the same. Results are discussed in relation to the calculated electrostatic potential minima and steric accessibility at the reactive site (1, 2).     

Radiolytic transformation of rotenone with potential anti-adipogenic activity

Radiolytic transformation of the isoflavonoid rotenone (1) with γ-irradiation afforded two new degraded products, rotenoisins A (2) and (3). The structures of the two new rotenone derivatives were elucidated on the basis of spectroscopic methods. The new products 2 and 3 exhibited significantly enhanced inhibitory activities against pancreatic lipase and adipocyte differentiation in 3T3-L1 cells when compared to parent rotenone.     

The effect of adriamycin on Z-DNA formation and DNA synthesis.

The ability of adriamycin to inhibit Z-DNA formation induced by a high-salt environment was investigated. ADM inhibited this conversion, such that in poly (dG-dC) total inhibition was observed at 1 ADM: 9 base pairs and in eukaryotic DNA (calf thymus) at 1 ADM: 11,5 base pairs. Even at low ADM concentration, 1 ADM: 160 base pairs, some inhibition was observed. At similar ADM:DNA concentrations, an inhibition in DNA synthesis in cells in culture was observed, which showed some parallel with the inhibition of Z-DNA formation. A model is proposed where Z-DNA formation precedes DNA synthesis and where inhibition of the former could explain the antineoplastic nature of adriamycin.     

Structural basis for Z-DNA binding and stabilization by the zebrafish Z-DNA dependent protein kinase PKZ

The RNA-dependent protein kinase PKR plays a central role in the antiviral defense of vertebrates by shutting down protein translation upon detection of viral dsRNA in the cytoplasm. In some teleost fish, PKZ, a homolog of PKR, performs the same function, but surprisingly, instead of dsRNA binding domains, it harbors two Z-DNA/Z-RNA-binding domains belonging to the Zalpha domain family. Zalpha domains have also been found in other proteins, which have key roles in the regulation of interferon responses such as ADAR1 and DNA-dependent activator of IFN-regulatory factors (DAI) and in viral proteins involved in immune response evasion such as the poxviral E3L and the Cyprinid Herpesvirus 3 ORF112. The underlying mechanism of nucleic acids binding and stabilization by Zalpha domains is still unclear. Here, we present two crystal structures of the zebrafish PKZ Zalpha domain (DrZalpha^PKZ) in alternatively organized complexes with a (CG)₆ DNA oligonucleotide at 2 and 1.8 Å resolution. These structures reveal novel aspects of the Zalpha interaction with DNA, and they give insights on the arrangement of multiple Zalpha domains on DNA helices longer than the minimal binding site.