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non-homologous end joining

Akihisa Takahashi, Eiichiro Mori, Yosuke Nakagawa, Atsuhisa Kajihara, Tadaaki Kirita, L Pittman Douglas, Masatoshi Hasegawa, Takeo Ohnishi
PURPOSE: Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock. MATERIALS AND METHODS: B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status...
October 24, 2016: International Journal of Hyperthermia
Elisabeth Bezine, Yann Malaisé, Aurore Loeuillet, Marianne Chevalier, Elisa Boutet-Robinet, Bernard Salles, Gladys Mirey, Julien Vignard
The Cytolethal Distending Toxin (CDT), produced by many bacteria, has been associated with various diseases including cancer. CDT induces DNA double-strand breaks (DSBs), leading to cell death or mutagenesis if misrepaired. At low doses of CDT, other DNA lesions precede replication-dependent DSB formation, implying that non-DSB repair mechanisms may contribute to CDT cell resistance. To address this question, we developed a proliferation assay using human cell lines specifically depleted in each of the main DNA repair pathways...
October 24, 2016: Scientific Reports
Fang Chen, Weifeng Zhang, Junli Zhao, Peiyan Yang, Rui Ma, Haibin Xia
Objective To prepare Rev-erbβ knockout HEK293 cells using clustered regularly interspaced short palindromic repeats/Cas 9 nuclease (CRISPR/Cas9) gene editing technology. Methods The knock-in or knockout of Rev-erbβ gene could be realized by single-guide RNA (sgRNA)-mediated Cas9 cutting of target DNA, and followed by DNA homologous recombination or non-homologous end joining-mediated DNA repair. Firstly, four sgRNAs were designed for Rev-erbβ gene. The sgRNA1 and sgRNA2 with the higher activity were respectively used to construct pCMV-hCas9-U6-Rev-erbβ sgRNA1 and pCMV-hCas9-U6-Rev-erbβ sgRNA2...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Zhangguo Chen, Katherine Gowan, Sonia M Leach, Sawanee S Viboolsittiseri, Ameet K Mishra, Tanya Kadoishi, Katrina Diener, Bifeng Gao, Kenneth Jones, Jing H Wang
BACKGROUND: Whole genome next generation sequencing (NGS) is increasingly employed to detect genomic rearrangements in cancer genomes, especially in lymphoid malignancies. We recently established a unique mouse model by specifically deleting a key non-homologous end-joining DNA repair gene, Xrcc4, and a cell cycle checkpoint gene, Trp53, in germinal center B cells. This mouse model spontaneously develops mature B cell lymphomas (termed G1XP lymphomas). RESULTS: Here, we attempt to employ whole genome NGS to identify novel structural rearrangements, in particular inter-chromosomal translocations (CTXs), in these G1XP lymphomas...
October 21, 2016: BMC Genomics
Kristin Bösch, Lamprinos Frantzeskakis, Miroslav Vraneš, Jörg Kämper, Kerstin Schipper, Vera Göhre
Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell's DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining...
September 30, 2016: Journal of Visualized Experiments: JoVE
James West, W Warren Gill
Genome editing in large animals has tremendous practical applications, from more accurate models for medical research through improved animal welfare and production efficiency. Although genetic modification in large animals has a 30 year history, until recently technical issues limited its utility. The original methods - pronuclear injection and integrating viruses - were plagued with problems associated with low efficiency, silencing, poor regulation of gene expression, and variability associated with random integration...
June 2016: Journal of Equine Veterinary Science
Jessica Brandi, Ilaria Dando, Elisa Dalla Pozza, Giulia Biondani, Rosalind Jenkins, Victoria Elliott, Kevin Park, Giuseppina Fanelli, Lello Zolla, Eithne Costello, Aldo Scarpa, Daniela Cecconi, Marta Palmieri
: Recently, we have shown that the secretome of pancreatic cancer stem cells (CSCs) is characterized by proteins that participate in cancer differentiation, invasion, and metastasis. However, the differentially expressed intracellular proteins that lead to the specific characteristics of pancreatic CSCs have not yet been identified, and as a consequence the deranged metabolic pathways are yet to be elucidated. To identify the modulated proteins of pancreatic CSCs, iTRAQ-based proteomic analysis was performed to compare the proteome of Panc1 CSCs and Panc1 parental cells, identifying 230 modulated proteins...
October 13, 2016: Journal of Proteomics
Julyun Oh, Amr Al-Zain, Elda Cannavo, Petr Cejka, Lorraine S Symington
The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex orchestrates the cellular response to DSBs through its structural, enzymatic, and signaling roles. Xrs2/Nbs1 is essential for nuclear translocation of Mre11, but its role as a component of the complex is not well defined. Here, we demonstrate that nuclear localization of Mre11 (Mre11-NLS) is able to bypass several functions of Xrs2, including DNA end resection, meiosis, hairpin resolution, and cellular resistance to clastogens. Using purified components, we show that the MR complex has equivalent activity to MRX in cleavage of protein-blocked DNA ends...
October 20, 2016: Molecular Cell
Jiawei Guan, Qian Zhao, Weifeng Mao
PTEN is a tumor suppressor gene characterized as a phosphatase that antagonizes the phosphatidylinositol 3-kinase signaling pathway in the cytoplasm. Nuclear PTEN plays roles in chromosomal stability, in which the double-strand breaks (DSB) repair mediated by homologous recombination (HR) and non-homologous end joining (NHEJ) is critical. Herein, the role of nuclear PTEN in DSB repair and the underlying molecular mechanism was investigated in this study. Using human breast cancer BT549 and MDA-MB-231 cell lines, we reveal a specific feature of PTEN that controls poly(ADP-ribosyl)ation of Ku70 and interferes with binding of Ku70 at DSB...
October 11, 2016: Biochimica et Biophysica Acta
Mae L Woods, Chris P Barnes
DNA double-strand breaks are lesions that form during metabolism, DNA replication and exposure to mutagens. When a double-strand break occurs one of a number of repair mechanisms is recruited, all of which have differing propensities for mutational events. Despite DNA repair being of crucial importance, the relative contribution of these mechanisms and their regulatory interactions remain to be fully elucidated. Understanding these mutational processes will have a profound impact on our knowledge of genomic instability, with implications across health, disease and evolution...
October 2016: PLoS Computational Biology
Wynand Paul Roos, Andrea Krumm
Histone/protein deacetylases play multiple roles in regulating gene expression and protein activation and stability. Their deregulation during cancer initiation and progression cause resistance to therapy. Here, we review the role of histone deacetylases (HDACs) and the NAD(+) dependent sirtuins (SIRTs) in the DNA damage response (DDR). These lysine deacetylases contribute to DNA repair by base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), homologous recombination (HR) and interstrand crosslink (ICL) repair...
October 13, 2016: Nucleic Acids Research
Ilenia Pellarin, Laura Arnoldo, Silvia Costantini, Silvia Pegoraro, Gloria Ros, Carlotta Penzo, Gianluca Triolo, Francesca Demarchi, Riccardo Sgarra, Alessandro Vindigni, Guidalberto Manfioletti
The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways...
2016: PloS One
Fatemeh Sanie-Jahromi, Iraj Saadat, Mostafa Saadat
AIMS: It has been shown that exposure to extremely-low frequency (˂300Hz) oscillating electromagnetic field (EMF) can affect gene expression. The effects of different exposure patterns of 50-Hz EMF and co-treatment of EMF plus cisplatin (CDDP) on mRNA levels of seven genes involved in DNA repair pathways (GADD45A, XRCC1, XRCC4, Ku70, Ku80, DNA-PKcs and LIG4) were evaluated. MAIN METHODS: Two 50-Hz EMF intensities (0.25 and 0.50mT), three exposure patterns (5min field-on/5min field-off, 15min field-on/15min field-off, 30min field-on continuously) and two cell lines (MCF-7 and SH-SY5Y) were used...
October 6, 2016: Life Sciences
Thomas Altmann, Andrew R Gennery
DNA ligase IV deficiency is a rare primary immunodeficiency, LIG4 syndrome, often associated with other systemic features. DNA ligase IV is part of the non-homologous end joining mechanism, required to repair DNA double stranded breaks. Ubiquitously expressed, it is required to prevent mutagenesis and apoptosis, which can result from DNA double strand breakage caused by intracellular events such as DNA replication and meiosis or extracellular events including damage by reactive oxygen species and ionising radiation...
October 7, 2016: Orphanet Journal of Rare Diseases
Johannes van den Boom, Markus Wolf, Lena Weimann, Nina Schulze, Fanghua Li, Farnusch Kaschani, Anne Riemer, Christian Zierhut, Markus Kaiser, George Iliakis, Hironori Funabiki, Hemmo Meyer
During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains...
October 6, 2016: Molecular Cell
Christos Coucoravas, Soniya Dhanjal, Sofia Henriksson, Stefanie Böhm, Marianne Farnebo
The cellular response to DNA double-strand breaks is orchestrated by the protein kinase ATM, which phosphorylates key actors in the DNA repair network. WRAP53β is a multifunctional protein that controls trafficking of factors to Cajal bodies, telomeres and DNA double-strand breaks but what regulates the involvement of WRAP53β in these separate processes remains unclear. Here, we show that in response to various types of DNA damage, including IR and UV, WRAP53β is phosphorylated on serine residue 64 by ATM with a time-course that parallels its accumulation at DNA lesions...
October 7, 2016: RNA Biology
Satish K Tadi, Carine Tellier-Lebègue, Clément Nemoz, Pascal Drevet, Stéphane Audebert, Sunetra Roy, Katheryn Meek, Jean-Baptiste Charbonnier, Mauro Modesti
In mammalian cells, classical non-homologous end joining (c-NHEJ) is critical for DNA double-strand break repair induced by ionizing radiation and during V(D)J recombination in developing B and T lymphocytes. Recently, PAXX was identified as a c-NHEJ core component. We report here that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components. PAXX-deficient cells display normal sensitivity to radiomimetic drugs, are proficient in transient V(D)J recombination assays, and do not shift toward higher micro-homology usage in plasmid repair assays...
October 4, 2016: Cell Reports
Anirban Chakraborty, Nisha Tapryal, Tatiana Venkova, Nobuo Horikoshi, Raj K Pandita, Altaf H Sarker, Partha S Sarkar, Tej K Pandita, Tapas K Hazra
DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes...
October 5, 2016: Nature Communications
Aiste McCormick, Peter Donoghue, Michelle Dixon, Richard O'Sullivan, Rachel Louise O'Donnell, James Murray, Angelika Kaufmann, Nicola J Curtin, Richard J Edmondson
PURPOSE: DNA damage defects are common in ovarian cancer and can be used to stratify treatment. Although most work has focussed on Homologous Recombination (HR), DNA double strand breaks are repaired primarily by non-homologous end joining (NHEJ). Defects in NHEJ have been shown to contribute to genomic instability and have been associated with the development of chemoresistance. EXPERIMENTAL DESIGN: NHEJ was assessed in a panel of ovarian cancer cell lines and 47 primary ascitic derived ovarian cancer cultures, by measuring the ability of cell extracts to end-join linearized plasmid monomers into multimers...
October 4, 2016: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Giulia Bastianello, Hiroshi Arakawa
All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system-gene conversion, somatic hypermutation and class switch recombination-require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 cell line, and conditionally expressed I-SceI endonuclease...
October 3, 2016: Nucleic Acids Research
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