double-strand break induction
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Gene targeting is becoming an important tool for precision genome engineering in plants. During gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. We have analysed gene targeting in barley (Hordeum vulgare) using a model system based on double-strand break (DSB) induction by the meganuclease I-SceI and a transgenic, artificial target locus. In the plants we obtained, the donor construct was inserted at the target locus by homology-directed DNA integration in at least two transformants obtained in a single experiment and was stably inherited as a single Mendelian trait. Both events were produced by one-sided integration. Our data suggest that gene replacement can be achieved in barley with a frequency suitable for routine application. The use of a codon-optimized nuclease and co-transfer of the nuclease gene together with the donor construct are probably the components important for efficient gene targeting. Such an approach, employing the recently developed synthetic nucleases/nickases that allow DSB induction at almost any sequence of a genome of interest, sets the stage for precision genome engineering as a routine tool even for important crops such as barley.
- Klíčová slova
- Barley, Hordeum vulgare, double-strand break induction, gene replacement, gene targeting, homology-directed DNA integration, precision genome engineering.,
- MeSH
- dvouřetězcové zlomy DNA * MeSH
- genetické lokusy MeSH
- geneticky modifikované rostliny MeSH
- genový targeting metody MeSH
- ječmen (rod) genetika MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- modely genetické MeSH
- reprodukovatelnost výsledků MeSH
- rostlinné geny MeSH
- transformace genetická MeSH
- typy dědičnosti genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Faithful chromosome segregation into gametes depends on Spo11-induced DNA double-strand breaks (DSBs). These yield single-stranded 3' tails upon resection to promote crossovers (COs). While early Mre11-dependent end resection is the predominant pathway in most organisms, Exo1 or Dna2/BLM can also contribute to the efficient processing of meiotic DSBs. Although its enzymatic activity has been thoroughly dissected, the temporal dynamics underlying Spo11 activity have remained mostly elusive. We show that, in Caenorhabditis elegans, SPO-11-mediated DSB induction takes place throughout early meiotic prophase I until mid-late pachynema. We find that late DSBs are essential for CO formation and are preferentially processed by EXO-1 and DNA-2 in a redundant fashion. Further, EXO-1-DNA-2-mediated resection ensures completion of conservative DSB repair and discourages activation of KU-dependent end joining. Taken together, our data unveil important temporal aspects of DSB induction and identify previously unknown functional implications for EXO-1-DNA-2-mediated resection activity in C. elegans.
- Klíčová slova
- CP: Cell biology, CP: Molecular biology,
- MeSH
- Caenorhabditis elegans * genetika metabolismus MeSH
- chromozomy metabolismus MeSH
- dvouřetězcové zlomy DNA MeSH
- meióza MeSH
- oprava DNA MeSH
- proteiny Caenorhabditis elegans * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- proteiny Caenorhabditis elegans * MeSH
DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.
- Klíčová slova
- CARM1, DNA repair, HDAC1, NBS1, PpoI, chromatin, nucleolus,
- MeSH
- acetylace MeSH
- arginin metabolismus MeSH
- buněčné jadérko genetika ultrastruktura MeSH
- buněčné linie MeSH
- dvouřetězcové zlomy DNA * MeSH
- embryonální kmenové buňky metabolismus ultrastruktura MeSH
- fosfoproteiny metabolismus MeSH
- geny rRNA MeSH
- histondeacetylasa 1 metabolismus MeSH
- histony metabolismus MeSH
- intracelulární signální peptidy a proteiny MeSH
- jaderné proteiny metabolismus MeSH
- metylace MeSH
- myši MeSH
- oprava DNA MeSH
- proteinarginin-N-methyltransferasy metabolismus MeSH
- RNA ribozomální genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- arginin MeSH
- coactivator-associated arginine methyltransferase 1 MeSH Prohlížeč
- fosfoproteiny MeSH
- Hdac1 protein, mouse MeSH Prohlížeč
- histondeacetylasa 1 MeSH
- histony MeSH
- intracelulární signální peptidy a proteiny MeSH
- jaderné proteiny MeSH
- proteinarginin-N-methyltransferasy MeSH
- RNA ribozomální MeSH
- Tcof1 protein, mouse MeSH Prohlížeč
Poly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in coordinating the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.
- MeSH
- buněčné jádro metabolismus MeSH
- Caenorhabditis elegans genetika metabolismus MeSH
- DNA metabolismus MeSH
- dvouřetězcové zlomy DNA * MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- oprava DNA fyziologie MeSH
- poly-ADP-ribosylace MeSH
- polyadenosindifosfátribosa metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- zárodečné buňky MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- DNA MeSH
- glykosidhydrolasy MeSH
- jaderné proteiny MeSH
- poly ADP-ribose glycohydrolase MeSH Prohlížeč
- polyadenosindifosfátribosa MeSH
- proteiny Caenorhabditis elegans MeSH
- SYP-1 protein, C elegans MeSH Prohlížeč
Most mitotic homologous recombination (HR) events proceed via a synthesis-dependent strand annealing mechanism to avoid crossing over, which may give rise to chromosomal rearrangements and loss of heterozygosity. The molecular mechanisms controlling HR sub-pathway choice are poorly understood. Here, we show that human RECQ5, a DNA helicase that can disrupt RAD51 nucleoprotein filaments, promotes formation of non-crossover products during DNA double-strand break-induced HR and counteracts the inhibitory effect of RAD51 on RAD52-mediated DNA annealing in vitro and in vivo. Moreover, we demonstrate that RECQ5 deficiency is associated with an increased occupancy of RAD51 at a double-strand break site, and it also causes an elevation of sister chromatid exchanges on inactivation of the Holliday junction dissolution pathway or on induction of a high load of DNA damage in the cell. Collectively, our findings suggest that RECQ5 acts during the post-synaptic phase of synthesis-dependent strand annealing to prevent formation of aberrant RAD51 filaments on the extended invading strand, thus limiting its channeling into potentially hazardous crossover pathway of HR.
- MeSH
- buněčné linie MeSH
- DNA opravný a rekombinační protein Rad52 metabolismus MeSH
- DNA metabolismus MeSH
- dvouřetězcové zlomy DNA * MeSH
- helikasy RecQ metabolismus MeSH
- jednovláknová DNA metabolismus MeSH
- lidé MeSH
- rekombinační oprava DNA * MeSH
- rekombinasa Rad51 metabolismus MeSH
- výměna sesterských chromatid MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA opravný a rekombinační protein Rad52 MeSH
- DNA MeSH
- helikasy RecQ MeSH
- jednovláknová DNA MeSH
- RECQL5 protein, human MeSH Prohlížeč
- rekombinasa Rad51 MeSH
Cell differentiation is associated with extensive gene silencing, heterochromatinization and potentially decreasing need for repairing DNA double-strand breaks (DSBs). Differentiation stages of blood cells thus represent an excellent model to study DSB induction, repair and misrepair in the context of changing higher-order chromatin structure. We show that immature granulocytes form γH2AX and 53BP1 foci, contrary to the mature cells; however, these foci colocalize only rarely and DSB repair is inefficient. Moreover, specific chromatin structure of granulocytes probably influences DSB induction.
- Klíčová slova
- Chromatin sensitivity to DSB induction, DNA double strand break (DSB) repair, Heterochromatin, Higher-order chromatin structure, Immature and terminally differentiated granulocytes, γH2AX/53BP1 repair foci,
- MeSH
- buněčná diferenciace * MeSH
- chromatin chemie MeSH
- hybridizace in situ fluorescenční MeSH
- konformace proteinů MeSH
- kultivované buňky MeSH
- lidé MeSH
- oprava DNA * MeSH
- poškození DNA * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chromatin MeSH
Survivin, as an antiapoptotic protein often overexpressed in cancer cells, is a logical target for potential cancer treatment. By overexpressing survivin, cancer cells can avoid apoptotic cell death and often become resistant to treatments, representing a significant obstacle in modern oncology. A survivin suppressor, an imidazolium-based compound known as YM-155, is nowadays studied as an attractive anticancer agent. Although survivin suppression by YM-155 is evident, researchers started to report that YM-155 is also an inducer of DNA damage introducing yet another anticancer mechanism of this drug. Moreover, the concentrations of YM-155 for DNA damage induction seems to be far lower than those needed for survivin inhibition. Understanding the molecular mechanism of action of YM-155 is of vital importance for modern personalized medicine involving the selection of responsive patients and possible treatment combinations. This review focuses mainly on the documented effects of YM-155 on DNA damage signaling pathways. It summarizes up to date literature, and it outlines the molecular mechanism of YM-155 action in the context of the DNA damage field.
- Klíčová slova
- DNA damage, YM-155, molecular mechanism of action, survivin,
- MeSH
- dvouřetězcové zlomy DNA účinky léků MeSH
- imidazoly farmakologie MeSH
- lidé MeSH
- naftochinony farmakologie MeSH
- poškození DNA účinky léků MeSH
- survivin metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- imidazoly MeSH
- naftochinony MeSH
- sepantronium MeSH Prohlížeč
- survivin MeSH
BACKGROUND: Human induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as γH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs). METHODS: We measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by γ-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. RESULTS: We demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages. CONCLUSIONS: Our observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.
- Klíčová slova
- 53BP1, DNA double-strand breaks, DNA repair, Human induced pluripotent stem cells, Long-term in vitro culture, γH2AX,
- MeSH
- 53BP1 genetika metabolismus MeSH
- buněčné linie MeSH
- DNA genetika metabolismus MeSH
- dvouřetězcové zlomy DNA * účinky záření MeSH
- exprese genu MeSH
- fibroblasty cytologie metabolismus účinky záření MeSH
- fosforylace účinky záření MeSH
- histony genetika metabolismus MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus účinky záření MeSH
- kontrolní body fáze G1 buněčného cyklu genetika MeSH
- kontrolní body fáze G2 buněčného cyklu genetika MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie metabolismus účinky záření MeSH
- oprava DNA genetika MeSH
- přeprogramování buněk MeSH
- stárnutí buněk genetika účinky záření MeSH
- záření gama MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 53BP1 MeSH
- DNA MeSH
- H2AX protein, human MeSH Prohlížeč
- histony MeSH
- TP53BP1 protein, human MeSH Prohlížeč
DNA double-strand breaks (DSBs), marked by ionizing radiation-induced (repair) foci (IRIFs), are the most serious DNA lesions and are dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold-standard method in radiation biodosimetry and allows research on DSB induction and repair at the molecular and single-cell levels. In this study, we introduce DeepFoci - a deep learning-based fully automatic method for IRIF counting and morphometric analysis. DeepFoci is designed to work with 3D multichannel data (trained for 53BP1 and γH2AX) and uses U-Net for nucleus segmentation and IRIF detection, together with maximally stable extremal region-based IRIF segmentation. The proposed method was trained and tested on challenging datasets consisting of mixtures of nonirradiated and irradiated cells of different types and IRIF characteristics - permanent cell lines (NHDFs, U-87) and primary cell cultures prepared from tumors and adjacent normal tissues of head and neck cancer patients. The cells were dosed with 0.5-8 Gy γ-rays and fixed at multiple (0-24 h) postirradiation times. Under all circumstances, DeepFoci quantified the number of IRIFs with the highest accuracy among current advanced algorithms. Moreover, while the detection error of DeepFoci remained comparable to the variability between two experienced experts, the software maintained its sensitivity and fidelity across dramatically different IRIF counts per nucleus. In addition, information was extracted on IRIF 3D morphometric features and repair protein colocalization within IRIFs. This approach allowed multiparameter IRIF categorization of single- or multichannel data, thereby refining the analysis of DSB repair processes and classification of patient tumors, with the potential to identify specific cell subclones. The developed software improves IRIF quantification for various practical applications (radiotherapy monitoring, biodosimetry, etc.) and opens the door to advanced DSB focus analysis and, in turn, a better understanding of (radiation-induced) DNA damage and repair.
- Klíčová slova
- 53BP1, P53-binding protein 1, Biodosimetry, CNN, convolutional neural network, Confocal Microscopy, Convolutional Neural Network, DNA Damage and Repair, DSB, DNA double-strand break, Deep Learning, FOV, field of view, GUI, graphical user interface, IRIF, ionizing radiation-induced (repair) foci, Image Analysis, Ionizing Radiation-Induced Foci (IRIFs), MSER, maximally stable extremal region (algorithm), Morphometry, NHDFs, normal human dermal fibroblasts, RAD51, DNA repair protein RAD51 homolog 1, U-87, U-87 glioblastoma cell line, γH2AX, histone H2AX phosphorylated at serine 139,
- Publikační typ
- časopisecké články MeSH
DNA double-strand breaks (DSBs), known as the most severe damage in chromatin, were induced in breast cancer cells and normal skin fibroblasts by 2 Gy ionizing photon radiation. In response to DSB induction, phosphorylation of the histone variant H2AX to γH2AX was observed in the form of foci visualized by specific antibodies. By means of super-resolution single-molecule localization microscopy (SMLM), it has been recently shown in a first article about these data that these foci can be separated into clusters of about the same size (diameter ~400 nm). The number of clusters increased with the dose applied and decreased with the repair time. It has also been shown that during the repair period, antibody-labeled MRE11 clusters of about half of the γH2AX cluster diameter were formed inside several γH2AX clusters. MRE11 is part of the MRE11-RAD50-NBS1 (MRN) complex, which is known as a DNA strand resection and broken-end bridging component in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). This article is a follow-up of the former ones applying novel procedures of mathematics (topology) and similarity measurements on the data set: to obtain a measure for cluster shape and shape similarities, topological quantifications employing persistent homology were calculated and compared. In addition, based on our findings that γH2AX clusters associated with heterochromatin show a high degree of similarity independently of dose and repair time, these earlier published topological analyses and similarity calculations comparing repair foci within individual cells were extended by topological data averaging (2nd-generation heatmaps) over all cells analyzed at a given repair time point; thereby, the two dimensions (0 and 1) expressed by components and holes were studied separately. Finally, these mean value heatmaps were averaged, in addition. For γH2AX clusters, in both normal fibroblast and MCF-7 cancer cell lines, an increased similarity was found at early time points (up to 60 min) after irradiation for both components and holes of clusters. In contrast, for MRE11, the peak in similarity was found at later time points (2 h up to 48 h) after irradiation. In general, the normal fibroblasts showed quicker phosphorylation of H2AX and recruitment of MRE11 to γH2AX clusters compared to breast cancer cells and a shorter time interval of increased similarity for γH2AX clusters. γH2AX foci and randomly distributed MRE11 molecules naturally occurring in non-irradiated control cells did not show any significant topological similarity.
