Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double-strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C-terminal domain, known as the SAP domain. Using single-particle cryo-electron microscopy, mass spectrometric analysis of intermolecular cross-linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA-bound state. The second position, which was observed in both apo and DNA-bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA. DATABASES: EM maps have been deposited in EMDB (EMD-11933). Coordinates have been deposited in Protein Data Bank (PDB 7AXZ). Other data are available from corresponding authors upon a request.
- MeSH
- antigen Ku chemie MeSH
- DNA chemie MeSH
- dvouřetězcové zlomy DNA * MeSH
- konformace proteinů MeSH
- lidé MeSH
- oprava DNA spojením konců * MeSH
- proteinové domény MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We report frequent losses of components of the classical nonhomologous end joining pathway (C-NHEJ), one of the main eukaryotic tools for end joining repair of DNA double-strand breaks, in several lineages of parasitic protists. Moreover, we have identified a single lineage among trypanosomatid flagellates that has lost Ku70 and Ku80, the core C-NHEJ components, and accumulated numerous insertions in many protein-coding genes. We propose a correlation between these two phenomena and discuss the possible impact of the C-NHEJ loss on genome evolution and transition to the parasitic lifestyle.IMPORTANCE Parasites tend to evolve small and compact genomes, generally endowed with a high mutation rate, compared with those of their free-living relatives. However, the mechanisms by which they achieve these features, independently in unrelated lineages, remain largely unknown. We argue that the loss of the classical nonhomologous end joining pathway components may be one of the crucial steps responsible for characteristic features of parasite genomes.
- MeSH
- antigen Ku chemie metabolismus MeSH
- Eukaryota metabolismus MeSH
- fylogeneze MeSH
- genom MeSH
- genomika metody MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- oprava DNA spojením konců * MeSH
- paraziti klasifikace genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raška, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.
- MeSH
- DNA-ligasa ATP genetika metabolismus MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- homologní rekombinace MeSH
- kultivované buňky MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie fyziologie MeSH
- nestabilita genomu * MeSH
- oprava DNA spojením konců fyziologie účinky záření MeSH
- oprava DNA fyziologie účinky záření MeSH
- proteiny vázající poly-ADP-ribosu genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non-homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ∼20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25-fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR-Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.
Methylation of histones H4 at lysine 20 position (H4K20me), which is functional in DNA repair, represents a binding site for the 53BP1 protein. Here, we show a radiation-induced increase in the level of H4K20me3 while the levels of H4K20me1 and H4K20me2 remained intact. H4K20me3 was significantly pronounced at DNA lesions in only the G1 phase of the cycle, while this histone mark was reduced in very late S and G2 phases when PCNA was recruited to locally micro-irradiated chromatin. H4K20me3 was diminished in locally irradiated Suv39h1/h2 double knockout (dn) fibroblasts, and the same phenomenon was observed for H3K9me3 and its binding partner, the HP1β protein. Immunoprecipitation showed the existence of an interaction between H3K9me3-53BP1 and H4K20me3-53BP1; however, HP1β did not interact with 53BP1. Together, H3K9me3 and H4K20me3 represent epigenetic markers that are important for the function of the 53BP1 protein in non-homologous end joining (NHEJ) repair. The very late S phase represents the cell cycle breakpoint when a DDR function of the H4K20me3-53BP1 complex is abrogated due to recruitment of the PCNA protein and other DNA repair factors of homologous recombination to DNA lesions.
- MeSH
- 53BP1 genetika metabolismus MeSH
- buněčné jádro genetika metabolismus účinky záření MeSH
- buněčné linie MeSH
- buněčný cyklus MeSH
- chromozomální proteiny, nehistonové metabolismus MeSH
- epigeneze genetická * účinky záření MeSH
- histony metabolismus MeSH
- lidé MeSH
- metylace DNA * účinky záření MeSH
- metylace MeSH
- myši MeSH
- oprava DNA spojením konců * MeSH
- poškození DNA * MeSH
- proliferační antigen buněčného jádra metabolismus MeSH
- restrukturace chromatinu MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chromatin DNA damage response (DDR) is orchestrated by the E3 ubiquitin ligase ring finger protein 168 (RNF168), resulting in ubiquitin-dependent recruitment of DDR factors and tumor suppressors breast cancer 1 (BRCA1) and p53 binding protein 1 (53BP1). This ubiquitin signaling regulates pathway choice for repair of DNA double-strand breaks (DSB), toxic lesions whose frequency increases during tumorigenesis. Recruitment of 53BP1 curbs DNA end resection, thereby limiting homologous recombination (HR) and directing DSB repair toward error-prone non-homologous end joining (NHEJ). Under cancer-associated ubiquitin starvation conditions reflecting endogenous or treatment-evoked proteotoxic stress, the ubiquitin-dependent accrual of 53BP1 and BRCA1 at the DNA damage sites is attenuated or lost. Challenging this current paradigm, here we identified diverse human cancer cell lines that display 53BP1 recruitment to DSB sites even under proteasome inhibitor-induced proteotoxic stress, that is, under substantial depletion of free ubiquitin. We show that central to this unexpected phenotype is overabundance of RNF168 that enables more efficient exploitation of the residual-free ubiquitin. Cells with elevated RNF168 are more resistant to combined treatment by ionizing radiation and proteasome inhibition, suggesting that such aberrant RNF168-mediated signaling might reflect adaptation to chronic proteotoxic and genotoxic stresses experienced by tumor cells. Moreover, the overabundant RNF168 and the ensuing unorthodox recruitment patterns of 53BP1, RIF1 and REV7 (monitored on laser micro-irradiation-induced DNA damage) shift the DSB repair balance from HR toward NHEJ, a scenario accompanied by enhanced chromosomal instability/micronuclei formation and sensitivity under replication stress-inducing treatments with camptothecin or poly(ADP-ribose) polymerase (PARP) inhibitor. Overall, our data suggest that the deregulated RNF168/53BP1 pathway could promote tumorigenesis by selecting for a more robust, better stress-adapted cancer cell phenotype, through altered DNA repair, fueling genomic instability and tumor heterogeneity. Apart from providing insights into cancer (patho)biology, the elevated RNF168, documented here also by immunohistochemistry on human clinical tumor specimens, may impact responses to standard-of-care and some emerging targeted cancer therapies.
- MeSH
- 53BP1 chemie genetika metabolismus MeSH
- aminokyselinové motivy MeSH
- fenotyp MeSH
- homeostáza účinky léků genetika MeSH
- karcinogeneze účinky léků genetika MeSH
- lidé MeSH
- mutace MeSH
- mutageny toxicita MeSH
- nádorové buněčné linie MeSH
- nestabilita genomu * účinky léků MeSH
- oprava DNA spojením konců účinky léků genetika MeSH
- oprava DNA účinky léků genetika MeSH
- poškození DNA MeSH
- regulace genové exprese u nádorů * MeSH
- signální transdukce účinky léků genetika MeSH
- transport proteinů účinky léků genetika MeSH
- ubikvitin metabolismus MeSH
- ubikvitinligasy genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Protein modifications regulate both DNA repair levels and pathway choice. How each modification achieves regulatory effects and how different modifications collaborate with each other are important questions to be answered. Here, we show that sumoylation regulates double-strand break repair partly by modifying the end resection factor Sae2. This modification is conserved from yeast to humans, and is induced by DNA damage. We mapped the sumoylation site of Sae2 to a single lysine in its self-association domain. Abolishing Sae2 sumoylation by mutating this lysine to arginine impaired Sae2 function in the processing and repair of multiple types of DNA breaks. We found that Sae2 sumoylation occurs independently of its phosphorylation, and the two modifications act in synergy to increase soluble forms of Sae2. We also provide evidence that sumoylation of the Sae2-binding nuclease, the Mre11-Rad50-Xrs2 complex, further increases end resection. These findings reveal a novel role for sumoylation in DNA repair by regulating the solubility of an end resection factor. They also show that collaboration between different modifications and among multiple substrates leads to a stronger biological effect.
- MeSH
- DNA vazebné proteiny genetika MeSH
- dvouřetězcové zlomy DNA MeSH
- endodeoxyribonukleasy genetika MeSH
- endonukleasy genetika MeSH
- exodeoxyribonukleasy genetika MeSH
- fosforylace MeSH
- lidé MeSH
- oprava DNA spojením konců genetika MeSH
- oprava DNA genetika MeSH
- poškození DNA genetika MeSH
- rozpustnost MeSH
- Saccharomyces cerevisiae - proteiny genetika MeSH
- Saccharomyces cerevisiae MeSH
- sumoylace genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Genome instability is associated with mitotic errors and cancer. This phenomenon can lead to deleterious rearrangements, but also genetic novelty, and many questions regarding its genesis, fate and evolutionary role remain unanswered. Here, we describe extreme chromosomal restructuring during genome elimination, a process resulting from hybridization of Arabidopsis plants expressing different centromere histones H3. Shattered chromosomes are formed from the genome of the haploid inducer, consistent with genomic catastrophes affecting a single, laggard chromosome compartmentalized within a micronucleus. Analysis of breakpoint junctions implicates breaks followed by repair through non-homologous end joining (NHEJ) or stalled fork repair. Furthermore, mutation of required NHEJ factor DNA Ligase 4 results in enhanced haploid recovery. Lastly, heritability and stability of a rearranged chromosome suggest a potential for enduring genomic novelty. These findings provide a tractable, natural system towards investigating the causes and mechanisms of complex genomic rearrangements similar to those associated with several human disorders.
- MeSH
- Arabidopsis genetika MeSH
- chromozomální aberace * MeSH
- cytogenetické vyšetření MeSH
- DNA primery genetika MeSH
- DNA-ligasy genetika MeSH
- genom rostlinný genetika MeSH
- genotyp MeSH
- hybridizace genetická genetika MeSH
- jednonukleotidový polymorfismus genetika MeSH
- molekulární sekvence - údaje MeSH
- nestabilita genomu genetika fyziologie MeSH
- oprava DNA spojením konců genetika MeSH
- poškození DNA genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In order to evaluate the ability of a representative polycyclic aromatic hydrocarbon (PAH) and PAH-containing complex mixtures to induce double strand DNA breaks (DSBs) and repair of damaged DNA in human embryonic lung fibroblasts (HEL12469 cells), we investigated the effect of benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5μm (PM2.5) on nonhomologous DNA end joining (NHEJ) and induction of stable chromosome aberrations (CAs). PM2.5 was collected in winter and summer 2011 in two Czech cities differing in levels and sources of air pollutants. The cells were treated for 24h with the following concentrations of tested chemicals: B[a]P: 1μM, 10μM, 25μM; EOMs: 1μg/ml, 10μg/ml, 25μg/ml. We tested several endpoints representing key steps leading from DSBs to the formation of CAs including histone H2AX phosphorylation, levels of proteins Ku70, Ku80 and XRCC4 participating in NHEJ, in vitro ligation activity of nuclear extracts of the HEL12469 cells and the frequency of stable CAs assessed by whole chromosome painting of chromosomes 1, 2, 4, 5, 7 and 17 using fluorescence in situ hybridization. Our results show that 25μM of B[a]P and most of the tested doses of EOMs induced DSBs as indicated by H2AX phosphorylation. DNA damage was accompanied by induction of XRCC4 expression and an increased frequency of CAs. Translocations most frequently affected chromosome 7. We observed only a weak induction of Ku70/80 expression as well as ligation activity of nuclear extracts. In summary, our data suggest the induction of DSBs and NHEJ after treatment of human embryonic lung fibroblasts with B[a]P and complex mixtures containing PAHs.
- MeSH
- antigeny jaderné genetika metabolismus MeSH
- benzopyren toxicita MeSH
- buněčné linie MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- embryo savčí metabolismus patologie MeSH
- fibroblasty metabolismus patologie MeSH
- fosforylace účinky léků genetika MeSH
- histony genetika metabolismus MeSH
- látky znečišťující životní prostředí toxicita MeSH
- lidé MeSH
- lidské chromozomy genetika metabolismus MeSH
- obnova měst MeSH
- oprava DNA spojením konců účinky léků genetika MeSH
- pevné částice MeSH
- plíce metabolismus patologie MeSH
- translokace genetická účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects. We will describe here a simple TALEN-based mutagenesis protocol suitable for the generation of germline mutations in Bombyx mori and Drosophila melanogaster. The protocol includes assembly of specific TAL modules, in vitro synthesis of TALEN RNAs, egg microinjection and mutation detection using PCR analysis. Our procedure allows a high frequency induction of NHEJ mutations, which often allows the reception of homozygous mutants already in the G1.
- MeSH
- bourec genetika MeSH
- deoxyribonukleasy genetika MeSH
- Drosophila melanogaster genetika MeSH
- dvouřetězcové zlomy DNA MeSH
- mikroinjekce přístrojové vybavení MeSH
- molekulární sekvence - údaje MeSH
- mutageneze cílená metody MeSH
- oprava DNA spojením konců * MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH