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.
- 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
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
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
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.
- 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
- 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
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.
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.
- 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
- práce podpořená grantem 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.
- 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
Double-strand breaks (DSBs), continuously introduced into DNA by cell metabolism, ionizing radiation and some chemicals, are the biologically most deleterious type of genome damage, and must be accurately repaired to protect genomic integrity, ensure cell survival, and prevent carcinogenesis. Although a huge amount of information has been published on the molecular basis and biological significance of DSB repair, our understanding of DSB repair and its spatiotemporal arrangement is still incomplete. In particular, the role of higher-order chromatin structure in DSB induction and repair, movement of DSBs and the mechanism giving rise to chromatin exchanges, and many other currently disputed questions are discussed in this review. Finally, a model explaining the formation of chromosome translocations is proposed.
We present new developments for an ab-initio model of the neutron relative biological effectiveness (RBE) in inducing specific classes of DNA damage. RBE is evaluated as a function of the incident neutron energy and of the depth inside a human-sized reference spherical phantom. The adopted mechanistic approach traces neutron RBE back to its origin, i.e. neutron physical interactions with biological tissues. To this aim, we combined the simulation of radiation transport through biological matter, performed with the Monte Carlo code PHITS, and the prediction of DNA damage using analytical formulas, which ground on a large database of biophysical radiation track structure simulations performed with the code PARTRAC. In particular, two classes of DNA damage were considered: sites and clusters of double-strand breaks (DSBs), which are known to be correlated with cell fate following radiation exposure. Within a coherent modelling framework, this approach tackles the variation of neutron RBE in a wide energy range, from thermal neutrons to neutrons of hundreds of GeV, and reproduces effects related to depth in the human-sized receptor, as well as to the receptor size itself. Besides providing a better mechanistic understanding of neutron biological effectiveness, the new model can support better-informed decisions for radiation protection: indeed, current neutron weighting (ICRP)/quality (U.S. NRC) factors might be insufficient for use in some radiation protection applications, because they do not account for depth. RBE predictions obtained with the reported model were successfully compared to the currently adopted radiation protection standards when the depth information is not relevant (at the shallowest depth in the phantom or for very high energy neutrons). However, our results demonstrate that great care is needed when applying weighting factors as a function of incident neutron energy only, not explicitly considering RBE variation in the target. Finally, to facilitate the use of our results, we propose look-up RBE tables, explicitly considering the depth variable, and an analytical representation of the maximal RBE vs. neutron energy.
