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1.
Int J Mol Sci ; 25(5)2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38473808

RESUMO

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.


Assuntos
DNA Forma Z , Animais , Humanos , Voluntários Saudáveis , Anticorpos Antinucleares , Imunoglobulina G , Imunoglobulina A
2.
Biochemistry ; 63(6): 777-787, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38437710

RESUMO

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.


Assuntos
DNA Forma Z , Ácidos Nucleicos , Conformação de Ácido Nucleico , Sítios de Ligação , RNA , Açúcares , Adenosina Desaminase/metabolismo
3.
J Am Chem Soc ; 146(11): 7584-7593, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38469801

RESUMO

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.


Assuntos
DNA Forma Z , Quimera de Direcionamento de Proteólise , Proteólise , Adenosina Desaminase/metabolismo , RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo
4.
Sci Rep ; 14(1): 4723, 2024 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-38413664

RESUMO

Z-DNA, a well-known non-canonical form of DNA involved in gene regulation, is often found in gene promoters. Transposable elements (TEs), which make up 45% of the human genome, can move from one location to another within the genome. TEs play various biological roles in host organisms, and like Z-DNA, can influence transcriptional regulation near promoter regions. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a critical role in the regulation of gene expression. Although TEs can generate Z-DNA and miRNAs can bind to Z-DNA, how these factors affect gene transcription has yet to be elucidated. Here, we identified potential Z-DNA forming sequence (ZFS), including TE-derived ZFS, in the promoter of prostaglandin reductase 1 (PTGR1) by data analysis. The transcriptional activity of these ZFS in PTGR1 was confirmed using dual-luciferase reporter assays. In addition, we discovered a novel ZFS-binding miRNA (miR-6867-5p) that suppressed PTGR1 expression by targeting to ZFS. In conclusion, these findings suggest that ZFS, including TE-derived ZFS, can regulate PTGR1 gene expression and that miR-6867-5p can suppress PTGR1 by interacting with ZFS.


Assuntos
DNA Forma Z , MicroRNAs , Humanos , Elementos de DNA Transponíveis/genética , Expressão Gênica , Regulação da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo
5.
Virus Res ; 343: 199342, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38408646

RESUMO

African swine fever virus is known to suppress type-I interferon (IFN) responses. The main objective of this study was to screen early-expressed viral genes for their ability to suppress IFN production. Out of 16 early genes examined, I73R exhibited robust suppression of cGAS-STING-induced IFN-ß promoter activities, impeding the function of both IRF3 and NF-κB transcription factors. As a result, I73R obstructed IRF3 nuclear translocation following the treatment of cells with poly(dA:dT), a strong inducer of the cGAS-STING signaling pathway. Although the I73R protein exhibits structural homology with the Zα domain binding to the left-handed helical form of DNA known as Z-DNA, its ability to suppress cGAS-STING induction of IFN-ß was independent of Z-DNA binding activity. Instead, the α3 and ß1 domains of I73R played a significant role in suppressing cGAS-STING induction of IFN-ß. These findings offer insights into the protein's functions and support its role as a virulence factor.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , DNA Forma Z , Interferon Tipo I , Animais , Suínos , Vírus da Febre Suína Africana/genética , Interferon beta/genética , Interferon beta/metabolismo , Transdução de Sinais/genética , Imunidade Inata/genética , DNA Forma Z/metabolismo , Proteínas de Membrana/metabolismo , Interferon Tipo I/metabolismo , Nucleotidiltransferases/genética
6.
Molecules ; 29(2)2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38276583

RESUMO

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.


Assuntos
DNA Forma Z , Quadruplex G , Água/química , DNA/química , Fenômenos Químicos , Conformação de Ácido Nucleico
7.
Nucleic Acids Res ; 52(4): 1575-1590, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38296834

RESUMO

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.


Assuntos
DNA Catalítico , DNA Forma Z , Quadruplex G , Animais , Camundongos , DNA Catalítico/metabolismo , Desoxirribonuclease I/metabolismo , Nuclease do Micrococo/genética , Cloreto de Sódio , Hemina , DNA Bacteriano/metabolismo , Biofilmes , Staphylococcus/genética , DNA , Polissacarídeos , Peroxidase/metabolismo , Mamíferos/genética
8.
Toxicol Appl Pharmacol ; 482: 116765, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37995810

RESUMO

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.


Assuntos
Antineoplásicos , Carbazóis , DNA Forma Z , Neoplasias Hepáticas , Humanos , Proteínas de Transporte/metabolismo , Necroptose , Proteínas Quinases/metabolismo , Apoptose , Antineoplásicos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Serina
9.
J Chem Theory Comput ; 20(2): 625-643, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38157247

RESUMO

Molecular dynamics simulations can be used in combination with experimental techniques to uncover the intricacies of biomolecular structure, dynamics, and the resulting interactions. However, many noncanonical nucleic acid structures have proven to be challenging to replicate in accurate agreement with experimental data, often attributed to known force field deficiencies. A common force field criticism is the handling of van der Waals (vdW) parameters, which have not been updated since the regular use of Ewald's methods became routine. This work dives into the effects of minute vdW radii shifts on RNA tetranucleotide, B-DNA, and Z-DNA model systems described by commonly used Amber force fields. Using multidimensional replica exchange molecular dynamics (M-REMD), the GACC RNA tetranucleotide demonstrated changes in the structural distribution between the NMR minor and anomalous structure populations based on the O2' vdW radii scanning. However, no significant change in the NMR Major conformation population was observed. There were minimal changes in the B-DNA structure but there were more substantial improvements in Z-DNA structural descriptions, specifically with the Tumuc1 force field. This occurred with both LJbb vdW radii adjustments and incorporation of the CUFIX nonbonded parameter modifications. Though the limited vdW modifications tested did not provide a universal fix to the challenge of simulating the various known nucleic acid structures, they do provide direction and a greater understanding for future force field development efforts.


Assuntos
DNA de Forma B , DNA Forma Z , Conformação de Ácido Nucleico , RNA/química , DNA , Simulação de Dinâmica Molecular
10.
Proc Natl Acad Sci U S A ; 121(2): e2308415120, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38150477

RESUMO

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.


Assuntos
Pareamento de Bases , Sistemas CRISPR-Cas , DNA Forma Z , Edição de Genes , Humanos , DNA/genética , DNA/química , DNA Forma Z/genética , Edição de Genes/métodos , RNA/genética , RNA Guia de Sistemas CRISPR-Cas , Timina/química
11.
J Am Chem Soc ; 146(1): 677-694, 2024 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-38131335

RESUMO

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.


Assuntos
DNA Forma Z , Ácidos Nucleicos , Conformação de Ácido Nucleico , Sítios de Ligação , DNA/química , RNA
12.
Immunity ; 56(11): 2508-2522.e6, 2023 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-37848037

RESUMO

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.


Assuntos
DNA de Forma B , DNA Forma Z , Espermina/metabolismo , Espermidina/metabolismo , DNA/metabolismo , Nucleotidiltransferases/metabolismo , Poliaminas/metabolismo , Imunidade Inata/genética
13.
Molecules ; 28(19)2023 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-37836806

RESUMO

RNA editing, a unique post-transcriptional modification, is observed in trypanosomatid parasites as a crucial procedure for the maturation of mitochondrial mRNAs. The editosome protein complex, involving multiple protein components, plays a key role in this process. In Trypanosoma brucei, a putative Z-DNA binding protein known as RBP7910 is associated with the editosome. However, the specific Z-DNA/Z-RNA binding activity and the interacting interface of RBP7910 have yet to be determined. In this study, we conducted a comparative analysis of the binding behavior of RBP7910 with different potential ligands using microscale thermophoresis (MST). Additionally, we generated a 3D model of the protein, revealing potential Z-α and Z-ß nucleic acid-binding domains of RBP7910. RBP7910 belongs to the winged-helix-turn-helix (HTH) superfamily of proteins with an α1α2α3ß1ß2 topology. Finally, using docking techniques, potential interacting surface regions of RBP7910 with notable oligonucleotide ligands were identified. Our findings indicate that RBP7910 exhibits a notable affinity for (CG)n Z-DNA, both in single-stranded and double-stranded forms. Moreover, we observed a broader interacting interface across its Z-α domain when bound to Z-DNA/Z-RNA compared to when bound to non-Z-form nucleic acid ligands.


Assuntos
DNA Forma Z , Trypanosoma brucei brucei , DNA Forma Z/metabolismo , RNA/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Edição de RNA , Citoplasma/metabolismo , Proteínas de Protozoários/química
14.
J Biol Chem ; 299(9): 105140, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37544644

RESUMO

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.


Assuntos
DNA , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA , Transcrição Gênica , Animais , DNA/química , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Forma Z/química , DNA Forma Z/genética , DNA Forma Z/metabolismo , Epigênese Genética , Genoma/genética , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , RNA/genética
15.
Genome Res ; 33(6): 907-922, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37433640

RESUMO

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.


Assuntos
DNA Forma Z , Nanoporos , Humanos , Motivos de Nucleotídeos , Análise de Sequência de DNA , DNA/genética , Composição de Bases , Sequenciamento de Nucleotídeos em Larga Escala
16.
Int J Mol Sci ; 24(13)2023 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-37445918

RESUMO

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, "bubbles", R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the "B-Z-topoII hypothesis" and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments ("Z-flipons") as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover "conformase" activity on given G(ate) B-DNA segments ("Z-flipins"), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural-functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents.


Assuntos
DNA de Forma B , DNA Forma Z , Humanos , Simulação de Acoplamento Molecular , DNA/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Guanosina Trifosfato , Adenosina Desaminase/metabolismo
17.
J Exp Med ; 220(8)2023 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-37450010

RESUMO

Nucleic acid sensing is a central process in the immune system, with far-reaching roles in antiviral defense, autoinflammation, and cancer. Z-DNA binding protein 1 (ZBP1) is a sensor for double-stranded DNA and RNA helices in the unusual left-handed Z conformation termed Z-DNA and Z-RNA. Recent research established ZBP1 as a key upstream regulator of cell death and proinflammatory signaling. Recognition of Z-DNA/RNA by ZBP1 promotes host resistance to viral infection but can also drive detrimental autoinflammation. Additionally, ZBP1 has interesting roles in cancer and other disease settings and is emerging as an attractive target for therapy.


Assuntos
DNA Forma Z , Ácidos Nucleicos , Proteínas de Ligação a RNA/metabolismo , Morte Celular , RNA/metabolismo
18.
Biomolecules ; 13(6)2023 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-37371544

RESUMO

Z-DNA refers to the left-handed double-helix DNA that has attracted much attention because of its association with some specific biological functions. However, because of its low content and unstable conformation, Z-DNA is normally difficult to observe or identify. Up to now, there has been a lack of unified or standard analytical methods among diverse techniques for probing Z-DNA and its transformation conveniently. In this work, NaCl, MgCl2, and ethanol were utilized to induce d(GC)8 from B-DNA to Z-DNA in vitro, and Fourier transform infrared (FTIR) spectroscopy was employed to monitor the transformation of Z-DNA under different induction conditions. The structural changes during the transformation process were carefully examined, and the DNA chirality alterations were validated by the circular dichroism (CD) measurements. The Z-DNA characteristic signals in the 1450 cm-1-900 cm-1 region of the d(GC)8 infrared (IR) spectrum were observed, which include the peaks at 1320 cm-1, 1125 cm-1 and 925 cm-1, respectively. The intensity ratios of A1320/A970, A1125/A970, and A925/A970 increased with Z-DNA content in the transition process. Furthermore, compared with the CD spectra, the IR spectra showed higher sensitivity to Z-DNA, providing more information about the molecular structure change of DNA. Therefore, this study has established a more reliable FTIR analytical approach to assess BZ DNA conformational changes in solutions, which may help the understanding of the Z-DNA transition mechanism and promote the study of Z-DNA functions in biological systems.


Assuntos
DNA Forma Z , Conformação de Ácido Nucleico , Espectrofotometria Infravermelho , DNA/química , Espectroscopia de Infravermelho com Transformada de Fourier , Dicroísmo Circular
19.
J Chem Theory Comput ; 19(13): 4299-4307, 2023 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-37340948

RESUMO

Advances in molecular dynamics (MD) software alongside enhanced computational power and hardware have allowed for MD simulations to significantly expand our knowledge of biomolecular structure, dynamics, and interactions. Furthermore, it has allowed for the extension of conformational sampling times from nanoseconds to the microsecond level and beyond. This has not only made convergence of conformational ensembles through comprehensive sampling possible but consequently exposed deficiencies and allowed the community to overcome limitations in the available force fields. The reproducibility and accuracy of the force fields are imperative in order to produce biologically relevant data. The Amber nucleic acid force fields have been used widely since the mid-1980s, and improvement of these force fields has been a community effort with several artifacts revealed, corrected, and reevaluated by various research groups. Here, we focus on the Amber force fields for use with double-stranded DNA and present the assessment of two recently developed force field parameter sets (OL21 and Tumuc1). Extensive MD simulations were performed with six test systems and two different water models. We observe the improvement of OL21 and Tumuc1 compared to previous generations of the Amber DNA force. We did not detect any significant improvement in the performance of Tumuc1 compared to OL21 despite the reparameterization of bonded force field terms in the former; however, we did note discrepancies in Tumuc1 when modeling Z-DNA sequences.


Assuntos
DNA Forma Z , DNA , Reprodutibilidade dos Testes , DNA/química , Simulação de Dinâmica Molecular , Conformação Molecular
20.
Life Sci Alliance ; 6(7)2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37164635

RESUMO

Identifying roles for Z-DNA remains challenging given their dynamic nature. Here, we perform genome-wide interrogation with the DNABERT transformer algorithm trained on experimentally identified Z-DNA forming sequences (Z-flipons). The algorithm yields large performance enhancements (F1 = 0.83) over existing approaches and implements computational mutagenesis to assess the effects of base substitution on Z-DNA formation. We show Z-flipons are enriched in promoters and telomeres, overlapping quantitative trait loci for RNA expression, RNA editing, splicing, and disease-associated variants. We cross-validate across a number of orthogonal databases and define BZ junction motifs. Surprisingly, many effects we delineate are likely mediated through Z-RNA formation. A shared Z-RNA motif is identified in SCARF2, SMAD1, and CACNA1 transcripts, whereas other motifs are present in noncoding RNAs. We provide evidence for a Z-RNA fold that promotes adaptive immunity through alternative splicing of KRAB domain zinc finger proteins. An analysis of OMIM and presumptive gnomAD loss-of-function datasets reveals an overlap of Z-flipons with disease-causing variants in 8.6% and 2.9% of Mendelian disease genes, respectively, greatly extending the range of phenotypes mapped to Z-flipons.


Assuntos
DNA Forma Z , RNA/genética , DNA/metabolismo , Genoma , Motivos de Nucleotídeos
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