Polyamine metabolism controls B-to-Z DNA transition to orchestrate DNA sensor cGAS activity.

Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) synthase (cGAS) is a universal double-stranded DNA (dsDNA) sensor that recognizes foreign and self-DNA in the cytoplasm and initiates innate immune responses and has been implicated in various infectious and non-infectious contexts. cGAS binds to the backbone of dsDNA and generates the second messenger, cGAMP, which activates the stimulator of interferon genes (STING). Here, we show that the endogenous polyamines spermine and spermidine attenuated cGAS activity and innate immune responses. Mechanistically, spermine and spermidine induced the transition of B-form DNA to Z-form DNA (Z-DNA), thereby decreasing its binding affinity with cGAS. Spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate-limiting enzyme in polyamine catabolism that decreases the cellular concentrations of spermine and spermidine, enhanced cGAS activation by inhibiting cellular Z-DNA accumulation; SAT1 deficiency promoted herpes simplex virus 1 (HSV-1) replication in vivo. The results indicate that spermine and spermidine induce dsDNA to adopt the Z-form conformation and that SAT1-mediated polyamine metabolism orchestrates cGAS activity.

AIRE relies on Z-DNA to flag gene targets for thymic T cell tolerization.

AIRE is an unconventional transcription factor that enhances the expression of thousands of genes in medullary thymic epithelial cells and promotes clonal deletion or phenotypic diversion of self-reactive T cells1-4. The biological logic of AIRE's target specificity remains largely unclear as, in contrast to many transcription factors, it does not bind to a particular DNA sequence motif. Here we implemented two orthogonal approaches to investigate AIRE's cis-regulatory mechanisms: construction of a convolutional neural network and leveraging natural genetic variation through analysis of F1 hybrid mice5. Both approaches nominated Z-DNA and NFE2-MAF as putative positive influences on AIRE's target choices. Genome-wide mapping studies revealed that Z-DNA-forming and NFE2L2-binding motifs were positively associated with the inherent ability of a gene's promoter to generate DNA double-stranded breaks, and promoters showing strong double-stranded break generation were more likely to enter a poised state with accessible chromatin and already-assembled transcriptional machinery. Consequently, AIRE preferentially targets genes with poised promoters. We propose a model in which Z-DNA anchors the AIRE-mediated transcriptional program by enhancing double-stranded break generation and promoter poising. Beyond resolving a long-standing mechanistic conundrum, these findings suggest routes for manipulating T cell tolerance.

Harnessing non-Watson-Crick's base pairing to enhance CRISPR effectors cleavage activities and enable gene editing in mammalian cells.

Genomic DNA of the cyanophage S-2L virus is composed of 2-aminoadenine (Z), thymine (T), guanine (G), and cytosine (C), forming the genetic alphabet ZTGC, which violates Watson-Crick base pairing rules. The Z-base has an extra amino group on the two position that allows the formation of a third hydrogen bond with thymine in DNA strands. Here, we explored and expanded applications of this non-Watson-Crick base pairing in protein expression and gene editing. Both ZTGC-DNA (Z-DNA) and ZUGC-RNA (Z-RNA) produced in vitro show detectable compatibility and can be decoded in mammalian cells, including Homo sapiens cells. Z-crRNA can guide CRISPR-effectors SpCas9 and LbCas12a to cleave specific DNA through non-Watson-Crick base pairing and boost cleavage activities compared to A-crRNA. Z-crRNA can also allow for efficient gene and base editing in human cells. Together, our results help pave the way for potential strategies for optimizing DNA or RNA payloads for gene editing therapeutics and give insights to understanding the natural Z-DNA genome.

Accurate sequencing of DNA motifs able to form alternative (non-B) structures.

Approximately 13% of the human genome at certain motifs have the potential to form noncanonical (non-B) DNA structures (e.g., G-quadruplexes, cruciforms, and Z-DNA), which regulate many cellular processes but also affect the activity of polymerases and helicases. Because sequencing technologies use these enzymes, they might possess increased errors at non-B structures. To evaluate this, we analyzed error rates, read depth, and base quality of Illumina, Pacific Biosciences (PacBio) HiFi, and Oxford Nanopore Technologies (ONT) sequencing at non-B motifs. All technologies showed altered sequencing success for most non-B motif types, although this could be owing to several factors, including structure formation, biased GC content, and the presence of homopolymers. Single-nucleotide mismatch errors had low biases in HiFi and ONT for all non-B motif types but were increased for G-quadruplexes and Z-DNA in all three technologies. Deletion errors were increased for all non-B types but Z-DNA in Illumina and HiFi, as well as only for G-quadruplexes in ONT. Insertion errors for non-B motifs were highly, moderately, and slightly elevated in Illumina, HiFi, and ONT, respectively. Additionally, we developed a probabilistic approach to determine the number of false positives at non-B motifs depending on sample size and variant frequency, and applied it to publicly available data sets (1000 Genomes, Simons Genome Diversity Project, and gnomAD). We conclude that elevated sequencing errors at non-B DNA motifs should be considered in low-read-depth studies (single-cell, ancient DNA, and pooled-sample population sequencing) and in scoring rare variants. Combining technologies should maximize sequencing accuracy in future studies of non-B DNA.

Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.

Z-RNA biology: a central role in the innate immune response?

Z-RNA is a higher-energy, left-handed conformation of RNA, whose function has remained elusive. A growing body of work alludes to regulatory roles for Z-RNA in the immune response. Here, we review how Z-RNA features present in cellular RNAs-especially containing retroelements-could be recognized by a family of winged helix proteins, with an impact on host defense. We also discuss how mutations to specific Z-contacting amino acids disrupt their ability to stabilize Z-RNA, resulting in functional losses. We end by highlighting knowledge gaps in the field, which, if addressed, would significantly advance this active area of research.

A fish herpesvirus highlights functional diversities among Zα domains related to phase separation induction and A-to-Z conversion.

Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains.

Zα Domain of ADAR1 Binds to an A-Form-like Nucleic Acid Duplex with Low Micromolar Affinity.

The left-handed Z-conformation of nucleic acids can be adopted by both DNA and RNA when bound by Zα domains found within a variety of viral and innate immune response proteins. While Z-form adoption is preferred by certain sequences, such as the commonly studied (CpG)n repeats, Zα has been reported to bind to a wide range of sequence contexts. Studying how Zα interacts with B-/A-form helices prior to their conversion to the Z-conformation is challenging as binding coincides with Z-form adoption. Here, we studied the binding of Zα fromHomo sapiens ADAR1 to a locked "A-type" version of the (CpG)3 construct (LNA (CpG)3) where the sugar pucker is locked into the C3'-endo/C2'-exo conformation, which prevents the duplex from adopting the alternating C2'/C3'-endo sugar puckers found in the Z-conformation. Using NMR and other biophysical techniques, we find that ZαADAR1 binds to the LNA (CpG)3 using a similar interface as for Z-form binding, with a dissociation constant (KD) of ∼4 µM. In contrast to Z-DNA/Z-RNA, where two ZαADAR1 bind to every 6 bp stretch, our data suggests that ZαADAR1 binds to multiple LNA molecules, indicating a completely different binding mode. Because ZαADAR1 binds relatively tightly to a non-Z-form model, its binding to B/A-form helices may need to be considered when experiments are carried out which attempt to identify the Z-form targets of Zα domains. The use of LNA constructs may be beneficial in experiments where negative controls for Z-form adoption are needed.

Flipons and small RNAs accentuate the asymmetries of pervasive transcription by the reset and sequence-specific microcoding of promoter conformation.

The role of alternate DNA conformations such as Z-DNA in the regulation of transcription is currently underappreciated. These structures are encoded by sequences called flipons, many of which are enriched in promoter and enhancer regions. Through a change in their conformation, flipons provide a tunable mechanism to mechanically reset promoters for the next round of transcription. They act as actuators that capture and release energy to ensure that the turnover of the proteins at promoters is optimized to cell state. Likewise, the single-stranded DNA formed as flipons cycle facilitates the docking of RNAs that are able to microcode promoter conformations and canalize the pervasive transcription commonly observed in metazoan genomes. The strand-specific nature of the interaction between RNA and DNA likely accounts for the known asymmetry of epigenetic marks present on the histone tetramers that pair to form nucleosomes. The role of these supercoil-dependent processes in promoter choice and transcriptional interference is reviewed. The evolutionary implications are examined: the resilience and canalization of flipon-dependent gene regulation is contrasted with the rapid adaptation enabled by the spread of flipon repeats throughout the genome. Overall, the current findings underscore the important role of flipons in modulating the readout of genetic information and how little we know about their biology.

Z-Form Adoption of Nucleic Acid is a Multi-Step Process Which Proceeds through a Melted Intermediate.

The left-handed Z-conformation of nucleic acids can be adopted by both DNA and RNA when bound by Zα domains found within a variety of innate immune response proteins. Zα domains stabilize this higher-energy conformation by making specific interactions with the unique geometry of Z-DNA/Z-RNA. However, the mechanism by which a right-handed helix contorts to become left-handed in the presence of proteins, including the intermediate steps involved, is poorly understood. Through a combination of nuclear magnetic resonance (NMR) and other biophysical measurements, we have determined that in the absence of Zα, under low salt conditions at room temperature, d(CpG) and r(CpG) constructs show no observable evidence of transient Z-conformations greater than 0.5% on either the intermediate or slow NMR time scales. At higher temperatures, we observed a transient unfolded intermediate. The ease of melting a nucleic acid duplex correlates with Z-form adoption rates in the presence of Zα. The largest contributing factor to the activation energies of Z-form adoption as calculated by Arrhenius plots is the ease of flipping the sugar pucker, as required for Z-DNA and Z-RNA. Together, these data validate the previously proposed "zipper model" for Z-form adoption in the presence of Zα. Overall, Z-conformations are more likely to be adopted by double-stranded DNA and RNA regions flanked by less stable regions and by RNAs experiencing torsional/mechanical stress.

Structurally Specific Z-DNA Proteolysis Targeting Chimera Enables Targeted Degradation of Adenosine Deaminase Acting on RNA 1.

Given the prevalent advancements in DNA- and RNA-based PROTACs, there remains a significant need for the exploration and expansion of more specific DNA-based tools, thus broadening the scope and repertoire of DNA-based PROTACs. Unlike conventional A- or B-form DNA, Z-form DNA is a configuration that exclusively manifests itself under specific stress conditions and with specific target sequences, which can be recognized by specific reader proteins, such as ADAR1 or ZBP1, to exert downstream biological functions. The core of our innovation lies in the strategic engagement of Z-form DNA with ADAR1 and its degradation is achieved by leveraging a VHL ligand conjugated to Z-form DNA to recruit the E3 ligase. This ingenious construct engendered a series of Z-PROTACs, which we utilized to selectively degrade the Z-DNA-binding protein ADAR1, a molecule that is frequently overexpressed in cancer cells. This meticulously orchestrated approach triggers a cascade of PANoptotic events, notably encompassing apoptosis and necroptosis, by mitigating the blocking effect of ADAR1 on ZBP1, particularly in cancer cells compared with normal cells. Moreover, the Z-PROTAC design exhibits a pronounced predilection for ADAR1, as opposed to other Z-DNA readers, such as ZBP1. As such, Z-PROTAC likely elicits a positive immunological response, subsequently leading to a synergistic augmentation of cancer cell death. In summary, the Z-DNA-based PROTAC (Z-PROTAC) approach introduces a modality generated by the conformational change from B- to Z-form DNA, which harnesses the structural specificity intrinsic to potentiate a selective degradation strategy. This methodology is an inspiring conduit for the advancement of PROTAC-based therapeutic modalities, underscoring its potential for selectivity within the therapeutic landscape of PROTACs to target undruggable proteins.

The role of DNA in the pathogenesis of SLE: DNA as a molecular chameleon.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterised by antibodies to DNA (anti-DNA) and other nuclear macromolecules. Anti-DNA antibodies are markers for classification and disease activity and promote pathogenesis by forming immune complexes that deposit in the tissue or stimulate cytokine production. Studies on the antibody response to DNA have focused primarily on a conformation of DNA known as B-DNA, the classic right-handed double helix. Among other conformations of DNA, Z-DNA is a left-handed helix with a zig-zag backbone; hence, the term Z-DNA. Z-DNA formation is favoured by certain base sequences, with the energetically unfavourable flip from B-DNA to Z-DNA dependent on conditions. Z-DNA differs from B-DNA in its immunogenicity in animal models. Furthermore, anti-Z-DNA antibodies, but not anti-B-DNA antibodies, can be present in otherwise healthy individuals. In SLE, antibodies to Z-DNA can occur in association with antibodies to B-DNA as a cross-reactive response, rising and falling together. While formed transiently in chromosomal DNA, Z-DNA is stably present in bacterial biofilms; biofilms can provide protection against antibiotics and other challenges including elements of host defence. The high GC content of certain bacterial DNA also favours Z-DNA formation as do DNA-binding proteins of bacterial or host origin. Together, these findings suggest that sources of Z-DNA can enhance the immunogenicity of DNA and, in SLE, stimulate the production of cross-reactive antibodies that bind both B-DNA and Z-DNA. As such, DNA can act as a molecular chameleon that, when stabilised in the Z-DNA conformation, can drive autoimmunity.

The anti-cancer drug candidate CBL0137 induced necroptosis via forming left-handed Z-DNA and its binding protein ZBP1 in liver cells.

CBL0137, a promising small molecular anti-cancer drug candidate, has been found to effectively induce apoptosis via activating p53 and suppressing nuclear factor-kappa B (NF-κB). However, it is still not clear whether CBL0137 can induce necroptosis in liver cancer; and if so, what is the underlying molecular mechanism. Here we found that CBL0137 could significantly induce left-handed double helix structure Z-DNA formation in HepG2 cells as shown by Z-DNA specific antibody assay, which was further confirmed by observing the expression of Z-DNA binding protein 1 (ZBP1) and adenosine deaminase acting on RNA 1 (ADAR1). Interestingly, we found that caspase inhibition significantly promoted CBL0137-induced necroptosis, which was further supported with the increase of the late apoptosis and necrosis assessed by the flow cytometry. Furthermore, we found that CBL0137 can also induce the expression of the three necroptosis-related proteins: receptor interacting serine/threonine kinase 1 (RIPK1), receptor interacting serine/threonine kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL). Taken together, it was assumed that CBL0137-indued necroptosis in liver cells was due to induction of Z-DNA and ZBP1, which activated RIPK1/RIPK3/MLKL pathway. This represents the first report on the induction of the Z-DNA-mediated necroptosis by CBL0137 in the liver cancer cells, which should provide new perspectives for CBL0137 treatment of liver cancer.

Inflammatory cell death PANoptosis is induced by the anti-cancer curaxin CBL0137 via eliciting the assembly of ZBP1-associated PANoptosome.

OBJECTIVE: PANoptosis, a new form of regulated cell death, concomitantly manifests hallmarks for pyroptosis, apoptosis, and necroptosis. It has been usually observed in macrophages, a class of widely distributed innate immune cells in various tissues, upon pathogenic infections. The second-generation curaxin, CBL0137, can trigger necroptosis and apoptosis in cancer-associated fibroblasts. This study aimed to explore whether CBL0137 induces PANoptosis in macrophages in vitro and in mouse tissues in vivo. METHODS: Bone marrow-derived macrophages and J774A.1 cells were treated with CBL0137 or its combination with LPS for indicated time periods. Cell death was assayed by propidium iodide staining and immunoblotting. Immunofluorescence microscopy was used to detect cellular protein distribution. Mice were administered with CBL0137 plus LPS and their serum and tissues were collected for biochemical and histopathological analyses, respectively. RESULTS: The results showed that CBL0137 alone or in combination with LPS induced time- and dose-dependent cell death in macrophages, which was inhibited by a combination of multiple forms of cell death inhibitors but not each alone. This cell death was independent of NLRP3 expression. CBL0137 or CBL0137 + LPS-induced cell death was characterized by simultaneously increased hallmarks for pyroptosis, apoptosis and necroptosis, indicating that this is PANoptosis. Induction of PANoptosis was associated with Z-DNA formation in the nucleus and likely assembly of PANoptosome. ZBP1 was critical in mediating CBL0137 + LPS-induced cell death likely by sensing Z-DNA. Moreover, intraperitoneal administration of CBL0137 plus LPS induced systemic inflammatory responses and caused multi-organ (including the liver, kidney and lung) injury in mice due to induction of PANoptosis in these organs. CONCLUSIONS: CBL0137 alone or plus inflammatory stimulation induces PANoptosis both in vitro and in vivo, which is associated with systemic inflammatory responses in mice.

Novel eGZ-motif formed by regularly extruded guanine bases in a left-handed Z-DNA helix as a major motif behind CGG trinucleotide repeats.

The expansion of d(CGG) trinucleotide repeats (TRs) lies behind several important neurodegenerative diseases. Atypical DNA secondary structures have been shown to trigger TR expansion: their characterization is important for a molecular understanding of TR disease. CD spectroscopy experiments in the last decade have unequivocally demonstrated that CGG runs adopt a left-handed Z-DNA conformation, whose features remain uncertain because it entails accommodating GG mismatches. In order to find this missing motif, we have carried out molecular dynamics (MD) simulations to explore all the possible Z-DNA helices that potentially form after the transition from B- to Z-DNA. Such helices combine either CpG or GpC Watson-Crick steps in Z-DNA form with GG-mismatch conformations set as either intrahelical or extrahelical; and participating in BZ or ZZ junctions or in alternately extruded conformations. Characterization of the stability and structural features (especially overall left-handedness, higher-temperature and steered MD simulations) identified two novel Z-DNA helices: the most stable one displays alternately extruded Gs, and is followed by a helix with symmetrically extruded ZZ junctions. The G-extrusion favors a seamless stacking of the Watson-Crick base pairs; extruded Gs favor syn conformations and display hydrogen-bonding and stacking interactions. Such conformations could have the potential to hijack the MMR complex, thus triggering further expansion.

Nonalternating purine pyrimidine sequences can form stable left-handed DNA duplex by strong topological constraint.

In vivo, left-handed DNA duplex (usually refers to Z-DNA) is mainly formed in the region of DNA with alternating purine pyrimidine (APP) sequence and plays significant biological roles. It is well known that d(CG)n sequence can form Z-DNA most easily under negative supercoil conditions, but its essence has not been well clarified. The study on sequence dependence of Z-DNA stability is very difficult without modification or inducers. Here, by the strong topological constraint caused by hybridization of two complementary short circular ssDNAs, left-handed duplex part was generated for various sequences, and their characteristics were investigated by using gel-shift after binding to specific proteins, CD and Tm analysis, and restriction enzyme cleavage. Under the strong topological constraint, non-APP sequences can also form left-handed DNA duplex as stable as that of APP sequences. As compared with non-APP sequences, the thermal stability difference for APP sequences between Z-form and B-form is smaller, which may be the reason that Z-DNA forms preferentially for APP ones. This result can help us to understand why nature selected APP sequences to regulate gene expression by transient Z-DNA formation, as well as why polymer with chirality can usually form both duplexes with left- or right-handed helix.

The Binding Properties of Antibodies to Z-DNA in the Sera of Normal Healthy Subjects.

Antibodies to DNA are a diverse set of antibodies that bind sites on DNA, a polymeric macromolecule that displays various conformations. In a previous study, we showed that sera of normal healthy subjects (NHS) contain IgG antibodies to Z-DNA, a left-handed helix with a zig-zig backbone. Recent studies have demonstrated the presence of Z-DNA in bacterial biofilms, suggesting a source of this conformation to induce responses. To characterize further antibodies to Z-DNA, we used an ELISA assay with brominated poly(dGdC) as a source of Z-DNA and determined the isotype of these antibodies and their binding properties. Results of these studies indicate that NHS sera contain IgM and IgA as well as IgG anti-Z-DNA antibodies. As shown by the effects of ionic strength in association and dissociation assays, the anti-Z-DNA antibodies bind primarily by electrostatic interactions; this type of binding differs from that of induced anti-Z-DNA antibodies from immunized animals which bind by non-ionic interactions. Furthermore, urea caused dissociation of NHS anti-Z-DNA at molar concentrations much lower than those for the induced antibodies. These studies also showed IgA anti-Z-DNA antibodies in fecal water. Together, these studies demonstrate that antibodies to Z-DNA occur commonly in normal immunity and may arise as a response to Z-DNA of bacterial origin.

On Water Arrangements in Right- and Left-Handed DNA Structures.

DNA requires hydration to maintain its structural integrity. Crystallographic analyses have enabled patterns of water arrangements to be visualized. We survey these water motifs in this review, focusing on left- and right-handed duplex and quadruplex DNAs, together with the i-motif. Common patterns of linear spines of water organization in grooves have been identified and are widely prevalent in right-handed duplexes and quadruplexes. By contrast, a left-handed quadruplex has a distinctive wheel of hydration populating the almost completely circular single groove in this structure.

Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind to nucleic acids.

Zα domains recognize the left-handed helical Z conformation of double-stranded nucleic acids. They are found in proteins involved in the nucleic acid sensory pathway of the vertebrate innate immune system and host evasion by viral pathogens. Previously, it has been demonstrated that ADAR1 (encoded by ADAR in humans) and DAI (also known as ZBP1) localize to cytoplasmic stress granules (SGs), and this localization is mediated by their Zα domains. To investigate the mechanism, we determined the interactions and localization pattern for the N-terminal region of human DAI (ZαßDAI), which harbours two Zα domains, and for a ZαßDAI mutant deficient in nucleic acid binding. Electrophoretic mobility shift assays demonstrated the ability of ZαßDAI to bind to hyperedited nucleic acids, which are enriched in SGs. Furthermore, using immunofluorescence and immunoprecipitation coupled with mass spectrometry, we identified several interacting partners of the ZαßDAI-RNA complex in vivo under conditions of arsenite-induced stress. These interactions are lost upon loss of nucleic acid-binding ability or upon RNase treatment. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to SGs is mainly mediated by the nucleic acid-binding ability of their Zα domains. This article has an associated First Person interview with Bharath Srinivasan, joint first author of the paper.

Thermodynamic analysis of Zα domain-nucleic acid interactions.

DNA/RNA molecules adopting the left-handed conformation (Z-form) have been attributed with immunogenic properties. However, their biological role and importance have been a topic of debate for many years. The discovery of Z-DNA/RNA binding domains (Zα domains) in varied proteins that are involved in the innate immune response, such as the interferon inducible form of the RNA editing enzyme ADAR1 (p150), Z-DNA binding protein 1 (ZBP1), the fish kinase PKZ and the poxvirus inhibitor of interferon response E3L, indicates important roles of Z-DNA/RNA in immunity and self/non-self-discrimination. Such Zα domain-containing proteins recognize left-handed Z-DNA/RNA in a conformation-specific manner. Recent studies have implicated these domains in virus recognition. Given these important emerging roles for the Zα domains, it is pivotal to understand the mechanism of recognition of the Z-DNA/Z-RNA by these domains. To this end, we assessed the binding thermodynamics of Zα domain from ORF112 and ADAR1 on T(CG)3 and T(CG)6 oligonucleotides which have high propensity to adopt the Z-conformation. Our study highlights important differences in the mode of oligonucleotide binding by the two Zα domains originating from different proteins. Site-directed mutagenesis was employed together with isothermal titration calorimetry to tease apart finer details of the binding thermodynamics. Our work advances the understanding on binding thermodynamics of Zα domains to their cognate nucleic acid substrates and paves the ground for future efforts to gain a complete appreciation of this process.