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G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.
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Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on the translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of noncanonical RNA:DNA hybrids in electrophoretic transport to the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a noncanonical helix, with distinct transport properties from B-form DD molecules. We find that RD and DD molecules, with the same contour length, move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes to be approximately the same. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and the molecular understanding of electrophoretic transport.
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DNA , Eletroforese , Simulação de Dinâmica Molecular , Nanoporos , RNA , RNA/química , DNA/química , Conformação de Ácido Nucleico , Nanotecnologia/métodosRESUMO
R-loops, composed of DNA-RNA hybrids and displaced single-stranded DNA, are known to pose a severe threat to genome integrity. Therefore, extensive research has focused on identifying regulatory proteins involved in controlling R-loop levels. These proteins play critical roles in preventing R-loop accumulation and associated genome instability. Herein I summarize recent knowledge on R-loop regulators affecting R-loop homeostasis, involving a wide array of R-loop screening methods that have enabled their characterization, from forward genetic and siRNA-based screens to proximity labeling and machine learning. These approaches not only deepen our understanding on R-loop formation processes, but also hold promise to find new targets in R-loop dysregulation associated with human pathologies.
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Estruturas R-Loop , Humanos , Instabilidade Genômica/genética , AnimaisRESUMO
Eukaryotic cells encounter diverse threats jeopardizing their integrity, prompting the development of defense mechanisms against these stressors. Among these mechanisms, inflammasomes are well-known for their roles in coordinating the inflammatory response against infections. Extensive research has unveiled their multifaceted involvement in cellular processes beyond inflammation. Recent studies emphasize the intricate relationship between the inflammasome and the DNA damage response (DDR). They highlight how the DDR participates in inflammasome activation and the reciprocal impact of inflammasome on DDR and genome integrity preservation. Moreover, novel functions of inflammasome sensors in DDR pathways have emerged, broadening our understanding of their roles. Finally, this review delves into identifying common signals that drive the activation of inflammasome sensors alongside activation cues for the DNA damage response, offering potential insights into shared regulatory pathways between these critical cellular processes.
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Transient state kinetic studies of eukaryotic DNA-dependent RNA polymerases (Pols) in vitro provide quantitative characterization of enzyme activity at the level of individual nucleotide addition events. Previous work revealed heterogeneity in the rate constants governing nucleotide addition by yeast RNA polymerase I (Pol I) for each position on a template DNA. In contrast, the rate constants that described nucleotide addition by yeast RNA polymerase II (Pol II) were more homogeneous. This observation led to the question, what drives the variability of rate constants governing RNA synthesis by Pol I? Are the kinetics of nucleotide addition dictated by the position of the nascent RNA within the polymerase or by the identity of the next encoded nucleotide? In this study, we examine the impact of nucleotide position (i.e. nascent RNA primer length) on the rate constants governing nine sequential nucleotide addition events catalyzed by Pol I. The results reveal a conserved trend in the observed rate constants at each position for all primer lengths used, and highlight that the 9-nucleotide, or 9-mer, RNA primer provides the fastest observed rate constants. These findings suggest that the observed heterogeneity of rate constants for RNA synthesis by Pol I in vitro is driven primarily by the template sequence.
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Nucleotídeos , RNA Polimerase I , RNA Polimerase I/metabolismo , Saccharomyces cerevisiae/metabolismo , Cinética , RNARESUMO
Flavonoids, low molecular weight polyphenolic compounds, are important natural products that belong to plant secondary metabolites. They have diverse biomedical applications such as antioxidative, anti-inflammatory, enzyme inhibitory, antimutagenic, anticarcinogenic, aromatase inhibitory effects, etc. Some of the flavonoids have been exported for bindings with certain DNA and tRNA structures both experimentally and computationally. RNA-DNA hybrid (RDH) falls into an important category of noncanonical nucleic acid structures that have many important biological functions. We have investigated the interaction of RDH structures with some of the dietary flavonoids with the aid of computational methods such as docking and molecular dynamics simulation. The presence of the - OH group on the ligand and the availability of a proper binding pocket in the macromolecule are the two main factors driving the binding preference. Thus, this computationally guided report explains the binding of the flavonoids with RDH structures to assist the researchers in designing noncanonical nucleic acid-targeted drug molecules.Communicated by Ramaswamy H. Sarma.
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t(8;14) translocation is the hallmark of Burkitt's lymphoma and results in c-MYC deregulation. During the translocation, c-MYC gene on chromosome 8 gets juxtaposed to the Ig switch regions on chromosome 14. Although the promoter of c-MYC has been investigated for its mechanism of fragility, little is known about other c-MYC breakpoint regions. We have analyzed the translocation break points at the exon 1/intron 1 of c-MYC locus from patients with Burkitt's lymphoma. Results showed that the breakpoint region, when present on a plasmid, could fold into an R-loop confirmation in a transcription-dependent manner. Sodium bisulfite modification assay revealed significant single-strandedness on chromosomal DNA of Burkitt's lymphoma cell line, Raji, and normal lymphocytes, revealing distinct R-loops covering up to 100 bp region. Besides, ChIP-DRIP analysis reveals that the R-loop antibody can bind to the breakpoint region. Further, we show the formation of stable parallel intramolecular G-quadruplex on non-template strand of the genome. Finally, incubation of purified AID in vitro or overexpression of AID within the cells led to enhanced mutation frequency at the c-MYC breakpoint region. Interestingly, anti-γH2AX can bind to DSBs generated at the c-MYC breakpoint region within the cells. The formation of R-loop and G-quadruplex was found to be mutually exclusive. Therefore, our results suggest that AID can bind to the single-stranded region of the R-loop and G4 DNA, leading to the deamination of cytosines to uracil and induction of DNA breaks in one of the DNA strands, leading to double-strand break, which could culminate in t(8;14) chromosomal translocation.
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Linfoma de Burkitt , Quadruplex G , Humanos , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , DNA , Genes myc , Estruturas R-Loop , Translocação GenéticaRESUMO
RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests that RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: They can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialized telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide colocalization with DNA breaks and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG and that mutation of RAD51 alters RNA-DNA hybrid abundance at 70 bp repeats both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.
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Trypanosoma brucei brucei , Estruturas R-Loop , Variação Antigênica/genética , Quebras de DNA , DNA , RNA , Glicoproteínas Variantes de Superfície de Trypanosoma/genéticaRESUMO
R-loops are unique, three-stranded nucleic acid structures that primarily form when an RNA molecule displaces one DNA strand and anneals to the complementary DNA strand in a double-stranded DNA molecule. R-loop formation can occur during natural processes, such as transcription, in which the nascent RNA molecule remains hybridized with the template DNA strand, while the non-template DNA strand is displaced. However, R-loops can also arise due to many non-natural processes, including DNA damage, dysregulation of RNA degradation pathways, and defects in RNA processing. Despite their prevalence throughout the whole genome, R-loops are predominantly found in actively transcribed gene regions, enabling R-loops to serve seemingly controversial roles. On one hand, the pathological accumulation of R-loops contributes to genome instability, a hallmark of cancer development that plays a role in tumorigenesis, cancer progression, and therapeutic resistance. On the other hand, R-loops play critical roles in regulating essential processes, such as gene expression, chromatin organization, class-switch recombination, mitochondrial DNA replication, and DNA repair. In this review, we summarize discoveries related to the formation, suppression, and removal of R-loops and their influence on genome instability, DNA repair, and oncogenic events. We have also discussed therapeutical opportunities by targeting pathological R-loops.
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With the development and wide usage of CRISPR technology, the presence of R-loop structures, which consist of an RNA-DNA hybrid and a displaced single-strand (ss) DNA, has become well accepted. R-loop structures have been implicated in a variety of circumstances and play critical roles in the metabolism of nucleic acid and relevant biological processes, including transcription, DNA repair, and telomere maintenance. Helicases are enzymes that use an ATP-driven motor force to unwind double-strand (ds) DNA, dsRNA, or RNA-DNA hybrids. Additionally, certain helicases have strand-annealing activity. Thus, helicases possess unique positions for R-loop biogenesis: they utilize their strand-annealing activity to promote the hybridization of RNA to DNA, leading to the formation of R-loops; conversely, they utilize their unwinding activity to separate RNA-DNA hybrids and resolve R-loops. Indeed, numerous helicases such as senataxin (SETX), Aquarius (AQR), WRN, BLM, RTEL1, PIF1, FANCM, ATRX (alpha-thalassemia/mental retardation, X-linked), CasDinG, and several DEAD/H-box proteins are reported to resolve R-loops; while other helicases, such as Cas3 and UPF1, are reported to stimulate R-loop formation. Moreover, helicases like DDX1, DDX17, and DHX9 have been identified in both R-loop formation and resolution. In this review, we will summarize the latest understandings regarding the roles of helicases in R-loop metabolism. Additionally, we will highlight challenges associated with drug discovery in the context of targeting these R-loop helicases.
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Estruturas R-Loop , RNA , DNA/metabolismo , Reparo do DNA , RNA/metabolismo , Humanos , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismoRESUMO
The senataxin (SETX, Sen1 in yeasts) RNA-DNA hybrid resolving helicase regulates multiple nuclear transactions, including DNA replication, transcription, and DNA repair, but the molecular basis for Sen1 activities is ill defined. Here, Sen1 cryoelectron microscopy (cryo-EM) reconstructions reveal an elongated inchworm-like architecture. Sen1 is composed of an amino terminal helical repeat Sen1 N-terminal (Sen1N) regulatory domain that is flexibly linked to its C-terminal SF1B helicase motor core (Sen1Hel) via an intrinsically disordered tether. In an autoinhibited state, the Sen1Sen1N domain regulates substrate engagement by promoting occlusion of the RNA substrate-binding cleft. The X-ray structure of an activated Sen1Hel engaging single-stranded RNA and ADP-SO4 shows that the enzyme encircles RNA and implicates a single-nucleotide power stroke in the Sen1 RNA translocation mechanism. Together, our data unveil dynamic protein-protein and protein-RNA interfaces underpinning helicase regulation and inactivation of human SETX activity by RNA-binding-deficient mutants in ataxia with oculomotor apraxia 2 neurodegenerative disease.
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Doenças Neurodegenerativas , RNA , Humanos , RNA/genética , Microscopia Crioeletrônica , RNA Helicases/genética , RNA Helicases/química , Enzimas Multifuncionais/genética , DNA/genética , Homeostase , DNA Helicases/genéticaRESUMO
The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. Here, we explored the role of FUS binding interactions for activity during transcription. In vitro run-off transcription assays revealed FUS-enhanced RNA produced by a non-eukaryote polymerase. The activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops from forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has the potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.
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DNA , Estruturas R-Loop , Proteína FUS de Ligação a RNA , RNA , DNA/metabolismo , Estruturas R-Loop/genética , RNA/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Ligação Proteica , Humanos , RNA Polimerases Dirigidas por DNA/metabolismo , Células HEK293RESUMO
Nucleic acid nanotechnology takes advantage of the self-assembling property of nucleic acids to form a variety of shapes and structures. The incorporation of metal ions into these structures introduces functionality for sensor and molecular electronic applications. Here, we describe a protocol for the incorporation of silver ions into polygonal nanoshapes that self-assemble from RNA and DNA modules.
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Nanoestruturas , Ácidos Nucleicos , Ácidos Nucleicos/química , Nanoestruturas/química , DNA/química , Nanotecnologia/métodos , Íons , Conformação de Ácido NucleicoRESUMO
Disruptions to the hemostatic pathway can cause a variety of serious or even life-threatening complications. Situations in which the coagulation of blood has become disturbed necessitate immediate care. Thrombin-binding aptamers are single-stranded nucleic acids that bind to thrombin with high specificity and affinity. While they can effectively inhibit thrombin, they suffer from rapid degradation and clearance in vivo. These issues are resolved, however, by attaching the therapeutic aptamer to a nucleic acid nanostructure. The increased size of the nanostructure-aptamer complex elongates the post-infusion half-life of the aptamer. These complexes are also immunoquiescent. A significant benefit of using nucleic acids as anticoagulants is their rapid deactivation by the introduction of a nanostructure made fully from the reverse complement of the therapeutically active nanostructure. These advantages make nanoparticle conjugated antithrombin aptamers a promising candidate for a rapidly reversible anticoagulant therapy.
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Aptâmeros de Nucleotídeos , Nanoestruturas , Trombina/metabolismo , RNA/farmacologia , Coagulação Sanguínea , Anticoagulantes/farmacologia , Anticoagulantes/química , Aptâmeros de Nucleotídeos/química , DNA/químicaRESUMO
Telomeric repeat containing RNA (TERRA) is transcribed from subtelomeric regions to telomeres. TERRA RNA can invade telomeric dsDNA and form telomeric R-loop structures. A growing body of evidence suggests that TERRA-mediated R-loops are critical players in telomere length homeostasis. Here, we will review current knowledge on the regulation of R-loop levels at telomeres. In particular, we will discuss how the central player TERRA and its binding proteins modulate R-loop levels through various mechanisms. We will further provide an overview of the consequences of TERRA-mediated persistent or unscheduled R-loops at telomeres in human ALT cancers and other organisms, with a focus on telomere length regulation after replication interference-induced damage and DNA homologous recombination-mediated repair.
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Nonhomologous end-joining (NHEJ) factors act in replication-fork protection, restart, and repair. Here, we identified a mechanism related to RNA:DNA hybrids to establish the NHEJ factor Ku-mediated barrier to nascent strand degradation in fission yeast. RNase H activities promote nascent strand degradation and replication restart, with a prominent role of RNase H2 in processing RNA:DNA hybrids to overcome the Ku barrier to nascent strand degradation. RNase H2 cooperates with the MRN-Ctp1 axis to sustain cell resistance to replication stress in a Ku-dependent manner. Mechanistically, the need of RNaseH2 in nascent strand degradation requires the primase activity that allows establishing the Ku barrier to Exo1, whereas impairing Okazaki fragment maturation reinforces the Ku barrier. Finally, replication stress induces Ku foci in a primase-dependent manner and favors Ku binding to RNA:DNA hybrids. We propose a function for the RNA:DNA hybrid originating from Okazaki fragments in controlling the Ku barrier specifying nuclease requirement to engage fork resection.
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RNA , Schizosaccharomyces , RNA/genética , RNA/metabolismo , DNA Primase/metabolismo , DNA/genética , DNA/metabolismo , Replicação do DNA , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Ribonucleases/genéticaRESUMO
The Development of reliable and field-compatible detection methods is essential to monitoring and controlling the spread of any global pandemic. We herein report a novel anti-RNA:DNA hybrid (anti-RDH) antibody-based biosensor for visual, colorimetric lateral flow assay, using gold nanoparticles, coupled with transcription-mediated-isothermal-RNA-amplification (TMIRA) for specific and sensitive detection of viral RNA. We have demonstrated its utility for SARS-CoV-2 RNA detection. This technique, which we have named RDH-LFA (anti-RNA:DNA hybrid antibody-based lateral flow assay), exploits anti-RDH antibody for immunocapture of viral RNA hybridized with specific DNA probes in lateral flow assay. This method uses biotinylated-oligonucleotides (DNAB) specific to SARS-CoV-2 RNA (vRNA) to generate a vRNA-DNAB hybrid. The biotin-tagged vRNA-DNAB hybrid molecules bind to streptavidin conjugated with gold nanoparticles. This hybrid complex is trapped by the anti-RDH antibody immobilized on the nitrocellulose membrane resulting in pink color signal leading to visual naked-eye detection in 1â minute. Combining RDH-LFA with isothermal RNA amplification (TMIRA) significantly improves the sensitivity (LOD:10â copies/µl) with a total turnaround time of an hour. More importantly, RDH-LFA coupled with the TMIRA method showed 96.6% sensitivity and 100% specificity for clinical samples when compared to a commercial gold standard reverse-transcription quantitative polymerase-chain-reaction assay. Thus, the present study reports a rapid, sensitive, specific, and simple method for visual detection of viral RNA, which can be used at the point-of-care without requiring sophisticated instrumentation.
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At critically short telomeres, stabilized TERRA RNA-DNA hybrids drive homology-directed repair (HDR) to delay replicative senescence. However, even at long- and intermediate-length telomeres, not subject to HDR, transient TERRA RNA-DNA hybrids form, suggestive of additional roles. We report that telomeric RNA-DNA hybrids prevent Exo1-mediated resection when telomeres become non-functional. We used the well-characterized cdc13-1 allele, where telomere resection can be induced in a temperature-dependent manner, to demonstrate that ssDNA generation at telomeres is either prevented or augmented when RNA-DNA hybrids are stabilized or destabilized, respectively. The viability of cdc13-1 cells is affected by the presence or absence of hybrids accordingly. Telomeric hybrids do not affect the shortening rate of bulk telomeres. We suggest that TERRA hybrids require dynamic regulation to drive HDR at short telomeres; hybrid presence may initiate HDR through replication stress, whereby their removal allows strand resection.
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RNA , Telômero , RNA/genética , Telômero/genética , DNA , Encurtamento do Telômero , DNA de Cadeia SimplesRESUMO
Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the replication of host chromosomes and how they operate. The initiator and replicator, proposed in the replicon hypothesis, are now well defined in both prokaryotes and eukaryotes. On the other hand, studies in prokaryotes and Archaea indicate alternative modes of initiation, which may not depend on an initiator. Here I summarize recent progress in the field of DNA replication and discuss the evolution of replication systems.
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Replicação do DNA , Origem de Replicação , Escherichia coli/metabolismo , Proteínas de Ligação a DNA/metabolismo , Replicon , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos , DNA Bacteriano/genéticaRESUMO
Nuclear RNA polymerase (Pol) III synthesizes large amounts of tRNAs and other short non-coding (nc)RNAs by a unique process that involves a termination-associated reinitiation-recycling mechanism. In addition to its two largest of 17 subunits, which contribute to active center RNA-DNA binding and catalytic site, a smaller subunit of ~110 aa (yeast C11, human RPC10) monitors this site, can modify its activity, and is essential for reinitiation-recycling. Distinct, but relevant to human immunity is cytoplasmic (cyto-)Pol III that is a direct sensor of AT-rich viral DNA from which it synthesizes 5'-ppp-RNA signaling molecules that activate interferon (IFN) production. Mutations in genes encoding Pol III subunits cause severe anti-viral immunodeficiency although the mechanisms responsible for cyto-Pol III initiation on this AT-rich DNA are unknown. Cyto-Pol III has also been implicated in inducing IFN in response to cytosolic mitochondrial DNA in autoimmune dysfunction. A focus of this commentary is recent biochemical and genetics research that examined the roles of the individual domains of C11 in the Pol III termination-associated reinitiation-recycling process as well as more recent cryo-EM structural and accompanying analyses, that are considered in evolutionary and other biological contexts. The N-terminal domain (NTD) of C11/RPC10 anchors at the periphery of Pol III from which a highly conserved linker extends to the mobile C-terminal RNA cleavage domain that can reach into the active center and rescue arrested complexes. Biochemical data indicate separable activities for the NTD and CTD in the transcription cycle, whereas the NTD-Linker can confer the evolutionary unique Pol III termination-reinitiation-recycling activity. A model produced from single particle cryo-EM conformations indicates that the C11-Linker-CTD swings in and out of the active center coordinated with allosteric movements of the DNA-binding clamp by the largest subunit, coupling termination to reinitiation-recycling. These may be relevant to DNA loading by cyto-Pol III during immune signaling.