DNA/RNA damage Dotaz Zobrazit nápovědu
RNA methylation, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we also observe that RNA modified at 8-methyladenosine (m8A) is recruited to UVA-damaged chromatin immediately after microirradiation. Interestingly, the level of m8A RNA at genomic lesions was reduced after inhibition of histone deacetylases and DNA methyltransferases. It appears in later phases of DNA damage response, accompanied by active DNA demethylation. Also, PARP inhibitor (PARPi), Olaparib, prevented adenosine methylation at microirradiated chromatin. PARPi abrogated not only m6A and m8A RNA positivity at genomic lesions, but also XRCC1, the factor of base excision repair (BER), did not recognize lesions in DNA. To this effect, Olaparib enhanced the genome-wide level of γH2AX. This histone modification interacted with m8A RNAs to a similar extent as m8A RNAs with DNA. Pronounced interaction properties we did not observe for m6A RNAs and DNA; however, m6A RNA interacted with XRCC1 with the highest efficiency, especially in microirradiated cells. Together, we show that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent. We suggest that modified RNAs likely play a role in the BER mechanism accompanied by active DNA demethylation. In this process, γH2AX stabilizes m6A/m8A-positive RNA-DNA hybrid loops via its interaction with m8A RNAs. R-loops could represent basic three-stranded structures recognized by PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.
The DNA damage response is mediated by both DNA repair proteins and epigenetic markers. Here, we observe that N6-methyladenosine (m6A), a mark of the epitranscriptome, was common in RNAs accumulated at UV-damaged chromatin; however, inhibitors of RNA polymerases I and II did not affect the m6A RNA level at the irradiated genomic regions. After genome injury, m6A RNAs either diffused to the damaged chromatin or appeared at the lesions enzymatically. DNA damage did not change the levels of METTL3 and METTL14 methyltransferases. In a subset of irradiated cells, only the METTL16 enzyme, responsible for m6A in non-coding RNAs as well as for splicing regulation, was recruited to microirradiated sites. Importantly, the levels of the studied splicing factors were not changed by UVA light. Overall, if the appearance of m6A RNAs at DNA lesions is regulated enzymatically, this process must be mediated via the coregulatory function of METTL-like enzymes. This event is additionally accompanied by radiation-induced depletion of 2,2,7-methylguanosine (m3G/TMG) in RNA. Moreover, UV-irradiation also decreases the global cellular level of N1-methyladenosine (m1A) in RNAs. Based on these results, we prefer a model in which m6A RNAs rapidly respond to radiation-induced stress and diffuse to the damaged sites. The level of both (m1A) RNAs and m3G/TMG in RNAs is reduced as a consequence of DNA damage, recognized by the nucleotide excision repair mechanism.
- MeSH
- adenosin analogy a deriváty metabolismus MeSH
- chromatin metabolismus MeSH
- demetylace DNA účinky záření MeSH
- fyziologický stres účinky záření MeSH
- guanosin analogy a deriváty metabolismus MeSH
- metylace DNA genetika účinky záření MeSH
- metylace účinky záření MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nekódující RNA metabolismus MeSH
- nestabilita genomu účinky záření MeSH
- poškození DNA MeSH
- RNA metabolismus MeSH
- ultrafialové záření * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Human cells are subjected to continuous challenges by different genotoxic stress attacks. DNA damage leads to erroneous mutations, which can alter the function of oncogenes or tumor suppressors, resulting in cancer development. To circumvent this, cells activate the DNA damage response (DDR), which mainly involves cell cycle regulation and DNA repair processes. The tumor suppressor p53 plays a pivotal role in the DDR by halting the cell cycle and facilitating the DNA repair processes. Various pathways and factors participating in the detection and repair of DNA have been described, including scores of RNA-binding proteins (RBPs) and RNAs. It has become increasingly clear that p53's role is multitasking, and p53 mRNA regulation plays a prominent part in the DDR. This review is aimed at covering the p53 RNA metabolism linked to the DDR and highlights the recent findings.
- MeSH
- lidé MeSH
- messenger RNA metabolismus MeSH
- mutace MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- nepřekládané oblasti MeSH
- oprava DNA * fyziologie MeSH
- poškození DNA * MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Úvod: Dlouhé nekódující ribonukleové kyseliny (long non-coding ribonucleic acids - lncRNA) jsou v poslední době vzhledem ke své úloze v procesu karcinogeneze předmětem zkoumání vědců zabývajících se nádory. Tyto transkripty regulují kritické kroky v normálních buněčných procesech, takže dysregulace jejich exprese se účastní patogeneze karcinomů. Z důvodu své blízkosti k lokusu CDKN1A má ncRNA spojená s P21 aktivovaná poškozením DNA (P21-associated ncRNA DNA damage activated - PANDA) v tomto ohledu zvláštní pozici. Podílí se na regulaci reakce na poškození DNA, stárnutí buněk a proliferace. Materiály a metody: V této studii jsme metodou kvantitativní polymerázové řetězové reakce hodnotili expresi této lncRNA ve tkáních karcinomu močového měchýře, sousedních nerakovinných tkání (adjacent non-cancerous tissues - ANCT) a v normálních vzorcích močového měchýře. Výsledky: Nebyl detekován žádný významný rozdíl v expresi PANDA, a to ani mezi nádorovými tkáněmi a ANCT (poměr exprese = 1,75; p = 0,11) nebo mezi nádorovými tkáněmi a normálními tkáněmi (poměr exprese = 2,72; p = 0,57). Úroveň exprese této lncRNA nebyla spojena s žádnými demografickými ani klinickými údaji o pacientech, jako je grade nádoru nebo recidiva, ani s rizikovými faktory souvisejícími s rakovinou, mezi něž patří např. kouření cigaret nebo závislost na opiu. Závěr: Tato studie tedy naznačuje, že PANDA není zapojena do patogeneze karcinomu močového měchýře. Hodnocení exprese jiných lncRNA by mohlo pomoci při identifikaci biomarkerů pro tyto karcinomy.
Background: Long non-coding RNAs (lncRNA) have recently been the focus of attention of cancer researchers due to their diverse roles in the carcinogenesis process. These transcripts regulate critical steps in the normal cellular processes, so dysregulation of their expression participate in the pathogenesis of several cancers. P21-associated ncRNA DNA damage activated (PANDA) has a special situation in this regard due to its adjacency to the CDKN1A locus. It is involved in the regulation of DNA damage response as well as cell senescence and proliferation. Material and methods: In the current study, we assessed the expression of this lncRNA in bladder cancer tissue, adjacent non- -cancerous tissues (ANCTs) and normal bladder samples by means of quantitative real time PCR method. Results: No significant difference has been detected in PANDA expression either between tumour tissue and ANCTs (expression ratio 1.75, P = 0.11) or between tumour tissue and normal tissues (expression ratio 2.72, P = 0.57). The expression level of this lncRNA was not associated with any of the demographic or clinical data of patients such as tumor grade or recurrence or cancer-associated risk factors such as cigarette smoking or opium addiction. Conclusion: Consequently, the current study implies that PANDA is not involved in the pathogenesis of bladder cancer. Assessment of expression of other lncRNAs would help in identification of biomarkers for this cancer.
- Klíčová slova
- ncRNA spojená s P21, aktivace poškozením DNA, PANDA,
- MeSH
- klinická studie jako téma MeSH
- lidé MeSH
- nádory močového měchýře * etiologie patologie MeSH
- nekódující RNA MeSH
- poškození DNA * MeSH
- RNA dlouhá nekódující MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate-dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21(Cip1) accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.
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- autoantigeny genetika metabolismus MeSH
- buněčný cyklus genetika MeSH
- CDC geny MeSH
- chromatin * genetika metabolismus MeSH
- chromozomy metabolismus MeSH
- DNA genetika metabolismus MeSH
- dvouřetězcové zlomy DNA MeSH
- fosfatasy cdc25 genetika metabolismus MeSH
- ionizující záření MeSH
- komplex Mi2-NuRD genetika metabolismus MeSH
- lidé MeSH
- malá interferující RNA metabolismus farmakologie MeSH
- nádorové buněčné linie MeSH
- oprava DNA * MeSH
- poškození DNA * fyziologie MeSH
- RNA interference MeSH
- signální transdukce * genetika MeSH
- ubikvitin genetika metabolismus MeSH
- ubikvitinace MeSH
- Check Tag
- lidé MeSH
Double-strand breaks (DSBs) are the most severe type of DNA damage. Previously, we demonstrated that RNA polymerase II (RNAPII) phosphorylated at the tyrosine 1 (Y1P) residue of its C-terminal domain (CTD) generates RNAs at DSBs. However, the regulation of transcription at DSBs remains enigmatic. Here, we show that the damage-activated tyrosine kinase c-Abl phosphorylates hSSB1, enabling its interaction with Y1P RNAPII at DSBs. Furthermore, the trimeric SOSS1 complex, consisting of hSSB1, INTS3, and c9orf80, binds to Y1P RNAPII in response to DNA damage in an R-loop-dependent manner. Specifically, hSSB1, as a part of the trimeric SOSS1 complex, exhibits a strong affinity for R-loops, even in the presence of replication protein A (RPA). Our in vitro and in vivo data reveal that the SOSS1 complex and RNAPII form dynamic liquid-like repair compartments at DSBs. Depletion of the SOSS1 complex impairs DNA repair, underscoring its biological role in the R-loop-dependent DNA damage response.
The cellular DNA damage response (DDR) machinery that maintains genomic integrity and prevents severe pathologies, including cancer, is orchestrated by signaling through protein modifications. Protein ubiquitylation regulates repair of DNA double-strand breaks (DSBs), toxic lesions caused by various metabolic as well as environmental insults such as ionizing radiation (IR). Whereas several components of the DSB-evoked ubiquitylation cascade have been identified, including RNF168 and BRCA1 ubiquitin ligases, whose genetic defects predispose to a syndrome mimicking ataxia-telangiectasia and cancer, respectively, the identity of the apical E1 enzyme involved in DDR has not been established. Here, we identify ubiquitin-activating enzyme UBA1 as the E1 enzyme required for responses to IR and replication stress in human cells. We show that siRNA-mediated knockdown of UBA1, but not of another UBA family member UBA6, impaired formation of both ubiquitin conjugates at the sites of DNA damage and IR-induced foci (IRIF) by the downstream components of the DSB response pathway, 53BP1 and BRCA1. Furthermore, chemical inhibition of UBA1 prevented IRIF formation and severely impaired DSB repair and formation of 53BP1 bodies in G 1, a marker of response to replication stress. In contrast, the upstream steps of DSB response, such as phosphorylation of histone H2AX and recruitment of MDC1, remained unaffected by UBA1 depletion. Overall, our data establish UBA1 as the apical enzyme critical for ubiquitylation-dependent signaling of both DSBs and replication stress in human cells, with implications for maintenance of genomic integrity, disease pathogenesis and cancer treatment.
- MeSH
- benzoáty farmakologie chemie MeSH
- buněčné jádro účinky léků MeSH
- dvouřetězcové zlomy DNA MeSH
- furany chemie farmakologie MeSH
- G1 fáze MeSH
- intracelulární signální peptidy a proteiny metabolismus MeSH
- ionizující záření MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- nádorové buněčné linie MeSH
- oprava DNA MeSH
- pyrazoly chemie farmakologie MeSH
- RNA interference MeSH
- ubikvitin aktivující enzymy antagonisté a inhibitory genetika metabolismus MeSH
- ubikvitinace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transposable elements (TEs) are powerful drivers of genome evolutionary dynamics but are principally deleterious to the host organism by compromising the integrity and function of the genome. The transposition of TEs may result in mutations and DNA damage. DNA double-strand breaks (DSBs), which may be caused by the transposition, are one of the processes directly linked to aging. TEs may thus be considered to constitute an internal source of aging and the frequency of transposition may, in turn, be considered to affect the pace of aging. The PIWI-piRNA pathway is a widespread strategy used by most animals to effectively suppress transposition. Interestingly, the PIWI-piRNA pathway is expressed predominantly in the animal germline, a more or less continuous immortal lineage set aside after the first few cell divisions of a developing embryo. Recent findings further imply that the PIWI-piRNA pathway and TE suppression constitute an important mechanism regulating aging. This article discusses the proposed role of the PIWI-piRNA pathway in setting the pace of aging as well as the possible mechanisms underlying this process.
- MeSH
- Argonaut proteiny * genetika metabolismus MeSH
- buněčné dělení genetika MeSH
- lidé MeSH
- malá interferující RNA * genetika metabolismus MeSH
- poškození DNA * MeSH
- stárnutí * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.
- MeSH
- fosforylace MeSH
- HeLa buňky MeSH
- histony metabolismus MeSH
- intracelulární signální peptidy a proteiny antagonisté a inhibitory chemie metabolismus MeSH
- kinetochory metabolismus MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- mitóza * MeSH
- nádorové buněčné linie MeSH
- oprava DNA * MeSH
- poškození DNA účinky záření MeSH
- protein-serin-threoninkinasy antagonisté a inhibitory genetika metabolismus MeSH
- proteinkinasa CDC2 metabolismus MeSH
- proteiny buněčného cyklu antagonisté a inhibitory genetika metabolismus MeSH
- protoonkogenní proteiny antagonisté a inhibitory genetika metabolismus MeSH
- RNA interference MeSH
- terciární struktura proteinů MeSH
- ubikvitinace MeSH
- záření gama MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([15N]5-8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5-8.9%, an inter-day precision of 3.4-8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer. Graphical abstract UHPLC-MS/MS analysis of DNA and RNA oxidative stress biomarkers.
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- biologické markery moč MeSH
- deoxyguanosin analogy a deriváty moč MeSH
- DNA moč MeSH
- dospělí MeSH
- extrakce na pevné fázi metody MeSH
- guanosin analogy a deriváty moč MeSH
- kreatin moč MeSH
- lidé MeSH
- limita detekce MeSH
- mladý dospělý MeSH
- nádory moč MeSH
- neurodegenerativní nemoci moč MeSH
- oxidační stres MeSH
- RNA moč MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
