«1. INTRODUCTION DNA is capable of assuming many different conformations other than the familiar right-handed B-DNA double helix . One of the most ...»
LEFT-HANDED Z-DNA: STRUCTURE AND
Alan Herbert and Alexander Rich
Department of Biology
Massachusetts Institute of Technology
77 Massachusetts Avenue
Cambridge, MA 02139
DNA is capable of assuming many different conformations other than the
familiar right-handed B-DNA double helix . One of the most dramatic
examples is the Z-DNA conformer, which is left-handed . Like B-DNA, the two strands of Z-DNA are antiparallel and joined by Watson-Crick base- pairing. In contrast to B-DNA, which has all its bases in the anti- conformation, the bases in the Z-DNA helix alternate between the anti conformation and the unusual syn conformation. This dinucleotide repeat causes the backbone to follow a zigzag path, giving rise to the name Z-DNA.
In Z-DNA there is only a single narrow groove that corresponds to the minor groove of B-DNA. No major groove exists. Instead, the "information" rich residues that allow sequence-specific recognition of B-DNA lie exposed on the convex outer surface of Z-DNA (Figure 1). This transition from B- to Z- DNA occurs most readily in sequences, with alternations of purines and pyrimidines, especially alternating deoxycytosine and deoxyguanine residues [3-5].
The biological role of Z-DNA is an area of active study. The aim of these investigations is to determine whether this alternate DNA conformations exist in vivo, how its formation is regulated, and what information it conveys. Here we will review recent studies that bear on the role of Z-DNA in biological systems.
1 2 Figure 1. The "information rich" residues that allow sequence specific recognition of the major groove of B-DNA lie on the convex surface of left-handed Z-DNA helix. The two DNA strands of each duplex are highlighted by solid black lines. The "zigzag" nature of the Z-DNA backbone is clearly seen (adapted from ).
2. FORMATION OF Z-DNA IN VITROThe existence of Z-DNA was first suggested by optical studies demonstrating that a polymer of alternating guanine and cytosine residues (d(CG)n) produced a nearly inverted circular dichroism spectrum in a high salt solution . The physical reason for this finding remained a mystery 3. 3 until an atomic resolution crystallographic study of d(CG)3 rather surprisingly revealed the existence of a left-handed double helix . Further experiments using Raman spectroscopy confirmed that the crystal structure was the same as formed when poly(d(CG)n) was placed in a high salt solution . Additional studies using circular dichroism to follow the transition from B- to Z-DNA demonstrated that Z-DNA can form from B- DNA under physiological salt conditions when deoxycytosine is 5- methylated . The subsequent discovery that Z-DNA formed under conditions of negative superhelical stress raised considerable excitement as this brought the left-handed conformation within the realm of biology [3, 5, 9].
Stabilization of Z-DNA by negative supercoiling illustrates a number of features about this conformation. First, formation of Z-DNA requires energy.
The amount necessary is proportional to the square of the number of negative supercoils lost from a covalently closed circular plasmid when a sequence fips into the Z-DNA conformation. For each turn of Z-DNA stabilized, approximately two supercoils are lost. The free energy required to effect the transition can be quantitated using two dimensional gel assays to follow the change in plasmid topology [10-14]. Second, sequences other than alternating purines and pyrimidines can form Z-DNA. The ease with which this occurs depends on the sequence - d(CG)n is best, d(TG)n is next, and a d(GGGC)n repeat is better than d(TA)n [12, 14, 15].Third, formation of B-Z DNA junctions, each of which has a free energy ∆G near +4 kcal/mole, is a significant energetic barrier to Z-DNA formation .
3. FORMATION OF Z-DNA IN VIVO
Due to the requirement for energy, formation of Z-DNA in vivo is an active process. One source of available energy is provided by transcription.
As pointed out by Liu and Wang, negative supercoils arise behind a moving RNA polymerase as it ploughs through the DNA double helix , providing one mechanism for the initiation of Z-DNA formation in vivo.
Computer models are consistent with this prediction. One analysis of 137 fully sequenced human genes demonstrated that sequences which could form Z-DNA easily were present in 98 genes. These sequences were distributed nonrandomly throughout a gene - sequences were ten times more frequent in 5' than in 3' regions . They lie precisely in the regions of a gene where negative supercoiling is highest during transcription.
4. EXPERIMENTAL DEMONSTRATION OF Z-DNA FORMATION IN VIVO
A number of experiments in prokaryotes have been used to demonstrate that Z-DNA forms in vivo, and that this occurs as a result of transcription.
One approach is to detect Z-DNA using chemical modification of DNA.
Through use of either osmium tetroxide or potassium permanganate, the formation within E. coli of Z-DNA in plasmids with a d(CG)n insert can be demonstrated [18, 19]. UV crosslinking of bacteria treated with psoralens have confirmed these results, and made possible a precise measurement of the amount of unrestrained supercoiling present within E. coli necessary to initiate formation of Z-DNA . Another approach has used a construct in which an EcoR1 site is embedded in a Z-DNA forming sequence [21-23]. In the bacterial cell, this fragment can be methylated when it is in the B-DNA conformation but it becomes resistant to methylation while in the Z-DNA conformation. Susceptibility to methylation of the EcoR1 site thus provides an in vivo measure of Z-DNA formation. In E. Coli, Z-DNA is formed in the absence of external perturbation and is increased by transcription, an effect that is enhanced by mutations inactivating topoisomerase I [22, 23]. In Morganella, Klebsiella, or Enterobacter formation of Z-DNA was not observed .
It has been difficult to directly demonstrate the existence of Z-DNA in eukaryotic systems due to their increased complexity. A number of early observations clearly suggested its existence. Unlike B-DNA, Z-DNA is highly immunogenic, and polyclonal as well as monoclonal antibodies can be made that recognize this conformation .One natural source rich in anti-Z-DNA antibodies is the sera obtained from patients with auto-immune diseases, especially lupus erythematosus . These antibodies are produced during the exacerbations of the disease, along with antibodies to many other nuclear components. The high specificity of these antibodies strongly suggest that Z-DNA is the cognate antigen, and by implication, that Z-DNA exists in vivo.
Antibodies raised in rabbits and sheep were used in staining experiments with both fixed  and unfixed polytene chromosomes of Drosophila .
These antibodies produced an unusual staining pattern of interband regions but did not stain bands. Staining was especially intense in the puffs, which are associated with high levels of transcriptional activity (reviewed in ).
Antibodies were also used in staining ciliated protozoa which have both a macronucleus and a micronucleus . The micronucleus is used for genetic reproduction, but the macronucleus is the site of all transcriptional activity.
Here, again, the macronucleus stained exclusively, with no staining in the 3. 5 micronucleus. Both of these early experiments suggested somewhat indirectly a link between transcriptional activity and the presence of Z-DNA.
Analysis of intact mammalian systems has been more complicated. There are a number of limitations in these experiments. As yet, no phenotype has been associated with presence or absence of Z-DNA forming sequences, thus excluding the use of genetic approaches. In order to model Z-DNA formation in vivo a number of experiments have been carried out using metabolically active permeabilized mammalian nuclei which were formed by embedding intact cells in agarose microbeads using the method of Jackson and Cook . Here, low concentrations of detergent are used to lyse the cytoplasmic membrane and permeabilize the nuclear membrane.
These nuclei have been shown to replicate DNA at 85% of the rate observed in the intact cell, and they are transcriptionally competent . In these experiments the amount of Z-DNA present in the gene is measured by diffusing biotin-labeled anti-Z-DNA monoclonal antibodies into the beads . The amount of Z-DNA present can be measured by quantitating how much radioactive streptavidin binds within the nucleus. Such experiments show that, at low concentrations of antibody, the amount of Z-DNA measured was independent of the antibody added over a 100-fold change in antibody concentration, suggesting that the Z-DNA is present de novo in these preparations rather than being induced by antibody. Furthermore, the amount of Z-DNA present increased dramatically during active transcription, consistent with the model of Liu and Wang , but was largely unaffected by DNA replication .
In further experiments, it was found that individual genes could be assayed by cross linking the antibody to DNA using a 10-nanosecond exposure of a laser at 266 nanometers . Release of DNA fragments with attached antibody was accomplished by diffusing in restriction endonucleases and performing an in situ DNA digest. Following isolation of biotin-labeled antibody-DNA complexes with streptavidin magnetobeads, free DNA was obtained by proteolysis. These experiments made it possible to determine the site of Z-DNA formation in particular genes. Using hybridization or PCR techniques, the c-myc gene was studied in murine U937 cells . Three transcription-dependent Z-DNA forming segments were identified in the 5' region of the gene with two of them near promoters . Retinoic acid, which induces the cells to differentiate into macrophages, was then used to down regulate expression of c-myc. Loss of c-myc expression was accompanied by a rapid reduction in the amount of ZDNA present in these three regions. In contrast, Z-DNA formation in the beta actin gene, which is not down regulated with differentiation, was detected under all the conditions tested.
6 In other studies with a primary liver cell line, induction of Z-DNA was measured in the corticotropin hormone-releasing gene . Z-DNA formation increased when the gene was up-regulated and decreased when it was down regulated. This finding suggests that physiological events are being measured in these systems.
A major conclusion from these studies is that Z-DNA forms largely, if not exclusively, behind a moving RNA polymerase and is stabilized by the negative supercoiling generated by DNA transcription.
5. FUNCTIONAL CONSEQUENCES OF Z-DNA FORMATION
The role of Z-DNA in biological processes is currently unknown. In principle, Z-DNA formation could have a functional role that need not involve its recognition by proteins. For example, E. coli RNA polymerase does not transcribe through Z-DNA  raising the possibility that the formation of Z-DNA behind (5') to a moving polymerase may block a trailing RNA polymerase from transcribing through that region of a gene until the torsional strain stabilizing the Z-DNA is relieved by topoisomerases. This mechanism might ensure spatial separation between successive polymerases. As a consequence, processing of an RNA would then be physically and temporally removed from that of subsequent transcripts, perhaps minimizing non-functional trans-splicing in eukaryotes.
Alternatively, formation of Z-DNA may relieve topological strain that arises when intact duplexes are intertwined as occurs during recombination events involving Holliday junction intermediates . For example, the Zforming d(CA/GT)n sequence has been shown to be recombinogenic in yeast , but is found to be less efficient than d(CG)n in human cells [40, 41].
Furthermore, several reports have correlated chromosomal breakpoints in human tumors to potential Z-DNA forming sequences, although no causal relationship has yet been established [42-46]. In addition, Z-DNA formation could affect the placement of nucleosomes as well as the organization of chromosomal domains by providing regions from which histones or other architectural proteins are excluded . Lastly, Z-DNA may perform unexpected roles in organisms such as the primitive eukaryote dinoflagellate Prorocentrum micans, which lack histones and nucleosomes but forms immunologically detectable Z-DNA at the nuclear periphery and at the segregation fork of dividing chromosomes .
There have been many attempts to find proteins that bind to Z-DNA in the hope that they would indicate indirectly the presence of Z-DNA in vivo, and help establish a biological role for this conformation. Early studies were 3. 7 unfruitful and caused widespread skepticism that Z-DNA would be associated with any biological function. Many of the apparently positive results reported in these studies may have been due either to artefacts or misinterpretation of data [49-51]. However, absence of proof was confused with absence of existence.
6. IDENTIFICATION OF A HIGH AFFINITY Z-DNA
BINDING PROTEIN WITH ENZYMATICACTIVITY Our work has recently shown that one type of double-stranded RNA adenosine deaminase (ADAR)  called ADAR1 binds Z-DNA in vitro with high affinity [53-55]. The dissociation constant of the Z-DNA binding domain is nanomolar, making it likely that this interaction is functional .
The binding of ADAR1 to Z-DNA was identified initially in bandshift assays, using competition with high concentrations of unlabeled polynucleotides to indirectly confirm specificity of binding . Mapping studies showed the presence in ADAR1 of two Z-DNA binding motifs, called Zα and Zβ  (Figure 2). Zα alone is able bind to Z-DNA with high affinity, but can interact with Zβ to form a domain with slightly different binding properties [57, 58]. The specificity of recombinant Zα for Z-DNA has now been directly confirmed using biophysical techniques such as circular dichroism and Raman spectroscopy [59, 60]. NMR studies have confirmed structure predictions that Zα belongs to the winged-helix- turnhelix family of proteins (Figure 3). The fold is similar to that found in the globular domain of histone H5  and the transcription factor HNF-γ3 .