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 crystal structure of the Zbeta domain of the RNA-editing enzyme ADAR1 reveals distinct conserved surfaces among Z-domains

The Zalpha domains represent a growing subfamily of the winged helix-turn-helix (HTH) domain family whose members share a remarkable ability to bind specifically to Z-DNA and/or Z-RNA. They have been found exclusively in proteins involved in interferon response and, while their importance in determining pox viral pathogenicity has been demonstrated, their actual target and biological role remain obscure. Cellular proteins containing Zalpha domains bear a second homologous domain termed Zbeta, which appears to lack the ability to bind left-handed nucleic acids. Here, we present the crystal structure of the Zbeta domain from the human double-stranded RNA adenosine deaminase ADAR1 at 0.97 A, determined by single isomorphous replacement including anomalous scattering. Zbeta maintains a winged-HTH fold with the addition of a C-terminal helix. Mapping of the Zbeta conservation profile on the Zbeta surface reveals a new conserved surface formed partly by the terminal helix 4, involved in metal binding and dimerization and absent from Zalpha domains. Our results show how two domains similar in fold may have evolved into different functional entities even in the context of the same protein.     

Zalpha-domains: at the intersection between RNA editing and innate immunity

The involvement of A to I RNA editing in antiviral responses was first indicated by the observation of genomic hyper-mutation for several RNA viruses in the course of persistent infections. However, in only a few cases an antiviral role was ever demonstrated and surprisingly, it turns out that ADARs - the RNA editing enzymes - may have a prominent pro-viral role through the modulation/down-regulation of the interferon response. A key role in this regulatory function of RNA editing is played by ADAR1, an interferon inducible RNA editing enzyme. A distinguishing feature of ADAR1, when compared with other ADARs, is the presence of a Z-DNA binding domain, Zalpha. Since the initial discovery of the specific and high affinity binding of Zalpha to CpG repeats in a left-handed helical conformation, other proteins, all related to the interferon response pathway, were shown to have similar domains throughout the vertebrate lineage. What is the biological function of this domain family remains unclear but a significant body of work provides pieces of a puzzle that points to an important role of Zalpha domains in the recognition of foreign nucleic acids in the cytoplasm by the innate immune system. Here we will provide an overview of our knowledge on ADAR1 function in interferon response with emphasis on Zalpha domains.     

Interaction of the Zalpha domain of human ADAR1 with a negatively supercoiled plasmid visualized by atomic force microscopy

Interest to the left-handed DNA conformation has been recently boosted by the findings that a number of proteins contain the Zα domain, which has been shown to specifically recognize Z-DNA. The biological function of Zα is presently unknown, but it has been suggested that it may specifically direct protein regions of Z-DNA induced by negative supercoiling in actively transcribing genes. Many studies, including a crystal structure in complex with Z-DNA, have focused on the human ADAR1 Zα domain in isolation. We have hypothesized that the recognition of a Z-DNA sequence by the Zα_ADAR1 domain is context specific, occurring under energetic conditions, which favor Z-DNA formation. To test this hypothesis, we have applied atomic force microscopy to image Zα_ADAR1 complexed with supercoiled plasmid DNAs. We have demonstrated that the Zα_ADAR1 binds specifically to Z-DNA and preferentially to d(CG)_n inserts, which require less energy for Z-DNA induction compared to other sequences. A notable finding is that site-specific Zα binding to d(GC)₁₃ or d(GC)₂C(GC)₁₀ inserts is observed when DNA supercoiling is insufficient to induce Z-DNA formation. These results indicate that Zα_ADAR1 binding facilities the B-to-Z transition and provides additional support to the model that Z-DNA binding proteins may regulate biological processes through structure-specific recognition.     

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.     

A left-handed RNA double helix bound by the Z alpha domain of the RNA-editing enzyme ADAR1

The A form RNA double helix can be transformed to a left-handed helix, called Z-RNA. Currently, little is known about the detailed structural features of Z-RNA or its involvement in cellular processes. The discovery that certain interferon-response proteins have domains that can stabilize Z-RNA as well as Z-DNA opens the way for the study of Z-RNA. Here, we present the 2.25 A crystal structure of the Zalpha domain of the RNA-editing enzyme ADAR1 (double-stranded RNA adenosine deaminase) complexed to a dUr(CG)(3) duplex RNA. The Z-RNA helix is associated with a unique solvent pattern that distinguishes it from the otherwise similar conformation of Z-DNA. Based on the structure, we propose a model suggesting how differences in solvation lead to two types of Z-RNA structures. The interaction of Zalpha with Z-RNA demonstrates how the interferon-induced isoform of ADAR1 could be targeted toward selected dsRNAs containing purine-pyrimidine repeats, possibly of viral origin.     

Biochemical characterization and preliminary X-ray crystallographic study of the domains of human ZBP1 bound to left-handed Z-DNA

ZBP1 is involved in host responses against cellular stresses, including tumorigenesis and viral infection. Structurally, it harbors two copies of the Zalpha domain containing the Zalpha motif, at its N terminus. Here, we attempted to characterize the Z-DNA binding activities of two Zalpha domains in the human ZBP1, hZalpha(ZBP1) and hZbeta(ZBP1), using circular dichroism (CD). Our results indicated that both hZalpha(ZBP1) and hZbeta(ZBP1) are viable Z-DNA binders, and their binding activities are comparable to those of previously-established Zalpha domains. Additionally, we crystallized hZbeta(ZBP1) in a complex with Z-DNA, d(TCGCGCG)2. The crystal diffracted to 1.45 angstroms, and belongs to the P2(1)2(1)2(1) space group, with the unit-cell parameters: a = 29.53 angstroms, b = 58.25 angstroms, and c = 88.61 angstroms. The delineation of this structure will provide insight into the manner in which diverse Zalpha motifs recognize Z-DNA.     

The zab domain of the human RNA editing enzyme ADAR1 recognizes Z-DNA when surrounded by B-DNA

The Zab domain of the editing enzyme ADAR1 binds tightly and specifically to Z-DNA stabilized by bromination or supercoiling. A stoichiometric amount of protein has been shown to convert a substrate of suitable sequence to the Z form, as demonstrated by a characteristic change in the CD spectrum of the DNA. Now we show that Zab can bind not only to isolated Z-forming d(CG)(n) sequences but also to d(CG)(n) embedded in B-DNA. The binding of Zab to such sequences results in a complex including Z-DNA, B-DNA, and two B-Z junctions. In this complex, the d(CG)(n) sequence, but not the flanking region, is in the Z conformation. The presence of Z-DNA was detected by cleavage with a Z-DNA specific nuclease, by undermethylation using Z-DNA sensitive SssI methylase, and by circular dichroism. It is possible that Zab binds to B-DNA with low affinity and flips any favorable sequence into Z-DNA, resulting in a high affinity complex. Alternatively, Zab may capture Z-DNA that exists transiently in solution. The binding of Zab to potential as well as established Z-DNA segments suggests that the range of biological substrates might be wider than previously thought.     

Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

Structure of the DLM-1-Z-DNA complex reveals a conserved family of Z-DNA-binding proteins

The first crystal structure of a protein, the Z alpha high affinity binding domain of the RNA editing enzyme ADAR1, bound to left-handed Z-DNA was recently described. The essential set of residues determined from this structure to be critical for Z-DNA recognition was used to search the database for other proteins with the potential for Z-DNA binding. We found that the tumor-associated protein DLM-1 contains a domain with remarkable sequence similarities to Z alpha(ADAR). Here we report the crystal structure of this DLM-1 domain bound to left-handed Z-DNA at 1.85 A resolution. Comparison of Z-DNA binding by DLM-1 and ADAR1 reveals a common structure-specific recognition core within the binding domain. However, the domains differ in certain residues peripheral to the protein-DNA interface. These structures reveal a general mechanism of Z-DNA recognition, suggesting the existence of a family of winged-helix proteins sharing a common Z-DNA binding motif.     

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.     

ADAR2 A-->I editing: site selectivity and editing efficiency are separate events

ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double-stranded. Site-selective A→I editing has been detected at specific sites within a few structured pre-mRNAs of metazoans. We have analyzed the editing selectivity of ADAR enzymes and have chosen to study the naturally edited R/G site in the pre-mRNA of the glutamate receptor subunit B (GluR-B). A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. Our results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double-stranded stem. To further understand the mechanism of selective ADAR2 editing we have investigated the importance of internal loops in the RNA substrate. We have found that the immediate structure surrounding the editing site is important. A purine opposite to the editing site has a negative effect on both selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affected editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events.     

Proteins that contain a functional Z-DNA-binding domain localize to cytoplasmic stress granules

Long double-stranded RNA may undergo hyper-editing by adenosine deaminases that act on RNA (ADARs), where up to 50% of adenosine residues may be converted to inosine. However, although numerous RNAs may undergo hyper-editing, the role for inosine-containing hyper-edited double-stranded RNA in cells is poorly understood. Nevertheless, editing plays a critical role in mammalian cells, as highlighted by the analysis of ADAR-null mutants. In particular, the long form of ADAR1 (ADAR1^p150) is essential for viability. Moreover, a number of studies have implicated ADAR1^p150 in various stress pathways. We have previously shown that ADAR1^p150 localized to cytoplasmic stress granules in HeLa cells following either oxidative or interferon-induced stress. Here, we show that the Z-DNA-binding domain (Zα^ADAR1) exclusively found in ADAR1^p150 is required for its localization to stress granules. Moreover, we show that fusion of Zα^ADAR1 to either green fluorescent protein (GFP) or polypyrimidine binding protein 4 (PTB4) also results in their localization to stress granules. We additionally show that the Zα domain from other Z-DNA-binding proteins (ZBP1, E3L) is likewise sufficient for localization to stress granules. Finally, we show that Z-RNA or Z-DNA binding is important for stress granule localization. We have thus identified a novel role for Z-DNA-binding domains in mammalian cells.