Poly(ADP-ribose) is synthesized by PARP enzymes during the repair of stochastic DNA breaks. Surprisingly, however, we show that most if not all endogenous poly(ADP-ribose) is detected in normal S phase cells at sites of DNA replication. This S phase poly(ADP-ribose) does not result from damaged or misincorporated nucleotides or from DNA replication stress. Rather, perturbation of the DNA replication proteins LIG1 or FEN1 increases S phase poly(ADP-ribose) more than 10-fold, implicating unligated Okazaki fragments as the source of S phase PARP activity. Indeed, S phase PARP activity is ablated by suppressing Okazaki fragment formation with emetine, a DNA replication inhibitor that selectively inhibits lagging strand synthesis. Importantly, PARP activation during DNA replication recruits the single-strand break repair protein XRCC1, and human cells lacking PARP activity and/or XRCC1 are hypersensitive to FEN1 perturbation. Collectively, our data indicate that PARP1 is a sensor of unligated Okazaki fragments during DNA replication and facilitates their repair.

• Klíčová slova
• DNA repair, DNA replication, DNA strand break, PARP1, postreplication repair,
• MeSH
• "flap" endonukleasy metabolismus   MeSH
• buněčné linie MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-ligasa ATP metabolismus   MeSH
• DNA genetika   metabolismus   MeSH
• lidé MeSH
• oprava DNA MeSH
• poly(ADP-ribosa)polymerasa 1 metabolismus   MeSH
• poly(ADP-ribosa)polymerasy genetika   metabolismus   MeSH
• polyadenosindifosfátribosa metabolismus   MeSH
• poškození DNA MeSH
• protein XRCC1 metabolismus   MeSH
• replikace DNA fyziologie   MeSH
• S fáze fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• "flap" endonukleasy MeSH
• DNA vazebné proteiny MeSH
• DNA-ligasa ATP MeSH
• DNA MeSH
• LIG1 protein, human MeSH Prohlížeč
• Okazaki fragments MeSH Prohlížeč
• poly(ADP-ribosa)polymerasa 1 MeSH
• poly(ADP-ribosa)polymerasy MeSH
• polyadenosindifosfátribosa MeSH
• protein XRCC1 MeSH

BACKGROUND: The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated. METHODS: The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells. RESULTS: This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm. CONCLUSION: The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.

• Klíčová slova
• DNA Damage Sensing, Genotoxic stress, MDM2, MRN complex, Precision medicine, RNA secondary structure, Synonymous mutations,
• MeSH
• ATM protein MeSH
• lidé MeSH
• nádorový supresorový protein p53 MeSH
• oprava DNA MeSH
• polynukleotid-5'-hydroxylkinasa * MeSH
• poškození DNA MeSH
• protoonkogenní proteiny c-mdm2 * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATM protein, human MeSH Prohlížeč
• ATM protein MeSH
• nádorový supresorový protein p53 MeSH
• polynukleotid-5'-hydroxylkinasa * MeSH
• protoonkogenní proteiny c-mdm2 * MeSH

We report a highly sensitive and selective multiplex assay by empowering an electrochemical DNA sensor with isothermal rolling circle amplification. The assay could simultaneously detect and discriminate three common entero-pathogens in a single reaction, with femtomolar sensitivity. It is useful for field- or resource-limited settings.

• MeSH
• biosenzitivní techniky * MeSH
• DNA genetika   MeSH
• elektrochemické techniky * MeSH
• Salmonella typhi izolace a purifikace   MeSH
• Shigella flexneri izolace a purifikace   MeSH
• techniky amplifikace nukleových kyselin * MeSH
• Vibrio cholerae izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH

We propose a label-free biosensor concept based on the charge state manipulation of nitrogen-vacancy (NV) quantum color centers in diamond, combined with an electrochemical microfluidic flow cell sensor, constructed on boron-doped diamond. This device can be set at a defined electrochemical potential, locking onto the particular chemical reaction, whilst the NV center provides the sensing function. The NV charge state occupation is initially prepared by applying a bias voltage on a gate electrode and then subsequently altered by exposure to detected charged molecules. We demonstrate the functionality of the device by performing label-free optical detection of DNA molecules. In this experiment, a monolayer of strongly cationic charged polymer polyethylenimine is used to shift the charge state of near surface NV centers from negatively charged NV- to neutral NV0 or dark positively charged NV+. Immobilization of negatively charged DNA molecules on the surface of the sensor restores the NV centers charge state back to the negatively charged NV-, which is detected using confocal photoluminescence microscopy. Biochemical reactions in the microfluidic channel are characterized by electrochemical impedance spectroscopy. The use of the developed electrochemical device can also be extended to nuclear magnetic resonance spin sensing.

• Klíčová slova
• DNA chip, biosensor, diamond, microfluidic, nitrogen-vacancy center,
• MeSH
• biosenzitivní techniky přístrojové vybavení   MeSH
• diamant chemie   MeSH
• DNA analýza   MeSH
• dusík chemie   MeSH
• elektrochemie MeSH
• laboratoř na čipu * MeSH
• polyethylenimin chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• diamant MeSH
• DNA MeSH
• dusík MeSH
• polyethylenimin MeSH

Real-time detection and nanoscale imaging of human immunodeficiency virus type 1 ribonucleic acid (HIV-1 RNA) in latently infected cells that persist in people living with HIV-1 on antiretroviral therapy in blood and tissue may reveal new insights needed to cure HIV-1 infection. Herein, we develop a strategy combining DNA nanotechnology and super-resolution expansion microscopy (ExM) to detect and image a 22 base sequence transcribed from the HIV-1 promoter in model live and fixed cells. We engineer a chimeric locked nucleic acid (LNA)-DNA sensor via hybridization chain reaction to probe HIV-1 RNA in the U3 region of the HIV-1 long terminal repeat (LTR) by signal amplification in live cells. We find that the viral RNA transcript of the U3 region of the HIV-1 LTR, namely PromA, is a valid and specific biomarker to detect infected live cells. The efficiency and selectivity of the LNA-DNA sensor are evaluated in combination with ExM. Unlike standard ExM methods, which rely on additional custom linkers to anchor and immobilize RNA molecules in the intracellular polymeric network, in the current strategy, we probe and image the HIV-1 RNA target at nanoscale resolution, without resorting to chemical linkers or additional preparation steps. This is achieved by physical entrapment of the HIV-1 viral transcripts in the cells post-expansion by finely tuning the mesh size of the intracellular polymeric network.

• MeSH
• DNA MeSH
• HIV-1 * genetika   MeSH
• lidé MeSH
• oligonukleotidy MeSH
• RNA virová genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• locked nucleic acid MeSH Prohlížeč
• oligonukleotidy MeSH
• RNA virová MeSH

Identifying grape varieties in wine, related products, and raw materials is of great interest for enology and to ensure its authenticity. However, these matrices' complexity and low DNA content make this analysis particularly challenging. Integrating DNA analysis with 2D materials, such as graphene, offers an advantageous pathway toward ultrasensitive DNA detection. Here, we show that monolayer graphene provides an optimal test bed for nucleic acid detection with single-base resolution. Graphene's ultrathinness creates a large surface area with quantum confinement in the perpendicular direction that, upon functionalization, provides multiple sites for DNA immobilization and efficient detection. Its highly conjugated electronic structure, high carrier mobility, zero-energy band gap with the associated gating effect, and chemical inertness explain graphene's superior performance. For the first time, we present a DNA-based analytic tool for grapevine varietal discrimination using an integrated portable biosensor based on a monolayer graphene field-effect transistor array. The system comprises a wafer-scale fabricated graphene chip operated under liquid gating and connected to a miniaturized electronic readout. The platform can distinguish closely related grapevine varieties, thanks to specific DNA probes immobilized on the sensor, demonstrating high specificity even for discriminating single-nucleotide polymorphisms, which is hard to achieve with a classical end-point polymerase chain reaction or quantitative polymerase chain reaction. The sensor was operated in ultralow DNA concentrations, with a dynamic range of 1 aM to 0.1 nM and an attomolar detection limit of ∼0.19 aM. The reported biosensor provides a promising way toward developing decentralized analytical tools for tracking wine authenticity at different points of the food value chain, enabling data transmission and contributing to the digitalization of the agro-food industry.

• Klíčová slova
• DNA sensor, SNP, field-effect transistor, grapevine, graphene, wine authenticity,
• MeSH
• biosenzitivní techniky * MeSH
• DNA sondy MeSH
• DNA chemie   MeSH
• grafit * chemie   MeSH
• polymerázová řetězová reakce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA sondy MeSH
• DNA MeSH
• grafit * MeSH

The mechanism by which DNA viruses interact with different DNA sensors and their connection with the activation of interferon (IFN) type I pathway are poorly understood. We investigated the roles of protein 204 (p204) and cyclic guanosine-adenosine synthetase (cGAS) sensors during infection with mouse polyomavirus (MPyV). The phosphorylation of IFN regulatory factor 3 (IRF3) and the stimulator of IFN genes (STING) proteins and the upregulation of IFN beta (IFN-β) and MX Dynamin Like GTPase 1 (MX-1) genes were detected at the time of replication of MPyV genomes in the nucleus. STING knockout abolished the IFN response. Infection with a mutant virus that exhibits defective nuclear entry via nucleopores and that accumulates in the cytoplasm confirmed that replication of viral genomes in the nucleus is required for IFN induction. The importance of both DNA sensors, p204 and cGAS, in MPyV-induced IFN response was demonstrated by downregulation of the IFN pathway observed in p204-knockdown and cGAS-knockout cells. Confocal microscopy revealed the colocalization of p204 with MPyV genomes in the nucleus. cGAS was found in the cytoplasm, colocalizing with viral DNA leaked from the nucleus and with DNA within micronucleus-like bodies, but also with the MPyV genomes in the nucleus. However, 2'3'-Cyclic guanosine monophosphate-adenosine monophosphate synthesized by cGAS was detected exclusively in the cytoplasm. Biochemical assays revealed no evidence of functional interaction between cGAS and p204 in the nucleus. Our results provide evidence for the complex interactions of MPyV and DNA sensors including the sensing of viral genomes in the nucleus by p204 and of leaked viral DNA and micronucleus-like bodies in the cytoplasm by cGAS.

• Klíčová slova
• cGAS sensor, immune sensing of DNA, mouse polyomavirus, p204 sensor, pattern recognition receptors,
• MeSH
• DNA virů genetika   imunologie   MeSH
• fosfoproteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• fosforylace MeSH
• infekce onkogenními viry imunologie   virologie   MeSH
• interakce hostitele a patogenu MeSH
• interferon beta metabolismus   MeSH
• jaderné proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• membránové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• myši MeSH
• nukleotidyltransferasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• polyomavirové infekce imunologie   virologie   MeSH
• Polyomavirus genetika   imunologie   MeSH
• přirozená imunita imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cGAS protein, mouse MeSH Prohlížeč
• DNA virů MeSH
• fosfoproteiny MeSH
• Ifi16 protein, mouse MeSH Prohlížeč
• interferon beta MeSH
• jaderné proteiny MeSH
• membránové proteiny MeSH
• nukleotidyltransferasy MeSH
• Sting1 protein, mouse MeSH Prohlížeč

DNA virus infections are often lifelong and can cause serious diseases in their hosts. Their recognition by the sensors of the innate immune system represents the front line of host defence. Understanding the molecular mechanisms of innate immunity responses is an important prerequisite for the design of effective antivirotics. This review focuses on the present state of knowledge surrounding the mechanisms of viral DNA genome sensing and the main induced pathways of innate immunity responses. The studies that have been performed to date indicate that herpesviruses, adenoviruses, and polyomaviruses are sensed by various DNA sensors. In non-immune cells, STING pathways have been shown to be activated by cGAS, IFI16, DDX41, or DNA-PK. The activation of TLR9 has mainly been described in pDCs and in other immune cells. Importantly, studies on herpesviruses have unveiled novel participants (BRCA1, H2B, or DNA-PK) in the IFI16 sensing pathway. Polyomavirus studies have revealed that, in addition to viral DNA, micronuclei are released into the cytosol due to genotoxic stress. Papillomaviruses, HBV, and HIV have been shown to evade DNA sensing by sophisticated intracellular trafficking, unique cell tropism, and viral or cellular protein actions that prevent or block DNA sensing. Further research is required to fully understand the interplay between viruses and DNA sensors.

• Klíčová slova
• DNA sensing, DNA viruses, IFI16, IFN, STING, TLR9, cGAS, inflammasome, innate immunity, p204/Ifi-204,
• MeSH
• DNA virů metabolismus   MeSH
• Herpesviridae * genetika   metabolismus   MeSH
• infekce DNA virem * MeSH
• lidé MeSH
• Polyomavirus * genetika   MeSH
• přirozená imunita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA virů MeSH

Lead contamination is now widespread, and exposure to lead may cause adverse effects on human beings. In this study, a photoelectrochemical sensor based on flower-like ZnO nanostructures was developed for Pb(2+) detection, using a Pb(2+)-dependent DNAzyme as the recognition unit and a double-strand DNA intercalator, Ru(bpy)2(dppz)(2+) (bpy=2,2'-bipyridine, dppz=dipyrido[3,2-a:2',3'-c] phenazine) as the photoelectrochemical signal reporter. The ZnO nanoflower was fabricated on an indium tin oxide (ITO) electrode by the convenient hydrothermal decomposition method. The morphology and photoelectrochemical property of the ZnO nanoflowers were characterized by SEM, XRD and photocurrent measurements. DNAzyme-substrate duplex was assembled on an ITO/ZnO electrode through electrostatic adsorption. In the presence of Pb(2+), RNA-cleavage activity of the DNAzyme was activated and its substrate strand was cleaved, resulting in the release of Ru(bpy)2(dppz)(2+) from the DNA film and the concomitant photocurrent decrease. The detection principle was verified by fluorescence measurements. Under the optimized conditions, a linear relationship between photocurrent and Pb(2+) concentration was obtained over the range of 0.5-20 nM, with a detection limit of 0.1 nM. Interference from other common metal ions was found negligible. Applicability of the sensor was demonstrated by analyzing lead level in human serum and Pb(2+) spiked water samples. This facile and economical sensor system showed high sensitivity and selectivity, thus can be potentially applied for on-site monitoring of lead contaminant.

• Klíčová slova
• DNAzyme, Lead contamination, Photoelectrochemical sensor, ZnO nanoflower,
• MeSH
• biosenzitivní techniky přístrojové vybavení   MeSH
• chemické látky znečišťující vodu analýza   MeSH
• design vybavení MeSH
• DNA katalytická chemie   MeSH
• elektrochemické techniky přístrojové vybavení   MeSH
• interkalátory chemie   MeSH
• jezera analýza   MeSH
• limita detekce MeSH
• nanostruktury chemie   ultrastruktura   MeSH
• olovo analýza   MeSH
• oxid zinečnatý chemie   MeSH
• pitná voda analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chemické látky znečišťující vodu MeSH
• DNA katalytická MeSH
• interkalátory MeSH
• olovo MeSH
• oxid zinečnatý MeSH
• pitná voda MeSH

High-throughput surface plasmon resonance (SPR) biosensor for rapid and parallelized detection of nucleic acids identifying specific bacterial pathogens is reported. The biosensor consists of a high-performance SPR imaging sensor with polarization contrast and internal referencing (refractive index resolution 2 x 10(-7) RIU) and an array of DNA probes microspotted on the surface of the SPR sensor. It is demonstrated that short sequences of nucleic acids (20-23 bases) characteristic for bacterial pathogens such as Brucella abortus, Escherichia coli, and Staphylococcus aureus can be detected at 100 pM levels. Detection of specific DNA or RNA sequences can be performed in less than 15 min by the reported SPR sensor.

• MeSH
• aerobní bakterie genetika   izolace a purifikace   MeSH
• analýza potravin metody   MeSH
• analýza selhání vybavení MeSH
• biosenzitivní techniky přístrojové vybavení   metody   MeSH
• design vybavení MeSH
• DNA bakterií analýza   genetika   MeSH
• kontaminace potravin analýza   MeSH
• potravinářská mikrobiologie * MeSH
• povrchová plasmonová rezonance přístrojové vybavení   metody   MeSH
• reprodukovatelnost výsledků MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů přístrojové vybavení   MeSH
• senzitivita a specificita MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH