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Alternative nhej

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
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
Valentina Palermo, Sara Rinalducci, Massimo Sanchez, Francesca Grillini, Joshua A Sommers, Robert M Brosh, Lello Zolla, Annapaola Franchitto, Pietro Pichierri
Regulation of end-processing is critical for accurate repair and to switch between homologous recombination (HR) and non-homologous end joining (NHEJ). End resection is a two-stage process but very little is known about regulation of the long-range resection, especially in humans. WRN participates in one of the two alternative long-range resection pathways mediated by DNA2 or EXO1. Here we demonstrate that phosphorylation of WRN by CDK1 is essential to perform DNA2-dependent end resection at replication-related DSBs, promoting HR, replication recovery and chromosome stability...
2016: Nature Communications
Ana López-Saavedra, Daniel Gómez-Cabello, María Salud Domínguez-Sánchez, Fernando Mejías-Navarro, María Jesús Fernández-Ávila, Christoffel Dinant, María Isabel Martínez-Macías, Jiri Bartek, Pablo Huertas
There are two major and alternative pathways to repair DNA double-strand breaks: non-homologous end-joining and homologous recombination. Here we identify and characterize novel factors involved in choosing between these pathways; in this study we took advantage of the SeeSaw Reporter, in which the repair of double-strand breaks by homology-independent or -dependent mechanisms is distinguished by the accumulation of green or red fluorescence, respectively. Using a genome-wide human esiRNA (endoribonuclease-prepared siRNA) library, we isolate genes that control the recombination/end-joining ratio...
2016: Nature Communications
Robin Sebastian, Sathees C Raghavan
Endosulfan (ES) is an organochlorine pesticide, speculated to be associated with chromosomal abnormalities and diseases in humans. However, very little is known about the mechanism of its genotoxicity. Using in vivo, ex vivo and in vitro model systems, we show that exposure to ES induces reactive oxygen species (ROS) in a concentration and time-dependent manner. The generation of ROS results in DNA double-strand breaks either directly or in a replication-dependent manner, both in mice and human cells. Importantly, ES-induced DNA damage evokes DNA damage response, resulting in elevated levels of classical non-homologous DNA endjoining (NHEJ), the predominant double-strand break repair pathway in higher eukaryotes...
October 2016: Carcinogenesis
Wei-Ping Lee, Ming-Chih Hou, Keng-Hsin Lan, Chung-Pin Li, Yee Chao, Han-Chieh Lin, Shou-Dong Lee
Helicobacter pylori infection leads to chronic gastritis and increased risk of gastric cancer. The mechanism involves chronic inflammation. We aimed to determine the mechanism by which H. pylori infection causes telomere shortening in inflammatory gastric mucosa. Gastric biopsy specimens were obtained from 20 patients with chronic gastritis or peptic ulcer caused by H. pylori infection. The specimens showed increased NF-κB and superoxide dismutase activities and elevated expressions of PARP-1 and γ-H2AX, all of which returned to normal levels after anti-H...
September 15, 2016: Archives of Biochemistry and Biophysics
Shigeaki Sunada, Hideki Kanai, Younghyun Lee, Takeshi Yasuda, Hirokazu Hirakawa, Cuihua Liu, Akira Fujimori, Mitsuru Uesaka, Ryuichi Okayasu
High-linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non-toxic concentration and carbon ions. NU7441-treated non-small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X-rays and carbon ions (290 MeV/n, 50 keV/μm)...
September 2016: Cancer Science
Charlene H Emerson, Alison A Bertuch
DNA double strand breaks (DSBs) are dangerous sources of genome instability and must be repaired by the cell. Nonhomologous end-joining (NHEJ) is an evolutionarily conserved pathway to repair DSBs by direct ligation of the ends, with no requirement for a homologous template. While NHEJ is the primary DSB repair pathway in mammalian cells, conservation of the core NHEJ factors throughout eukaryotes makes the pathway attractive for study in model organisms. The budding yeast, Saccharomyces cerevisiae, has been used extensively to develop a functional picture of NHEJ...
March 31, 2016: Biochemistry and Cell Biology, Biochimie et Biologie Cellulaire
Kai Yang, Rong Guo, Dongyi Xu
DNA double-strand breaks (DSBs) are the most serious form of DNA damage. In human cells, non-homologous end joining (NHEJ) is the major pathway for the repair of DSBs. Different types of DSBs result in different subsets of NHEJ repair strategies. These variations in NHEJ repair strategies depend on numerous elements, such as the flexible recruitment of NHEJ-related proteins, the complexity of the DSB ends, and the spatial- and temporal-ordered formation of the multi-protein complex. On the one hand, current studies of DNA DSBs repair focus on the repair pathway choices between homologous recombination and classic or alternative NHEJ...
July 2016: Acta Biochimica et Biophysica Sinica
Janapriya Saha, Anthony J Davis
Heritable mutations in the tumor suppressor gene BRCA1 increase a woman's lifetime risk of developing breast and ovarian cancer. BRCA1's tumor suppressor function is directly linked to its myriad of functions in the cellular response to DNA double-strand breaks (DSBs). BRCA1 interacts with an extensive array of DNA damage responsive proteins and plays important roles in DSB repair, mediated by the homologous recombination pathway, and in the activation of cell cycle checkpoints. However, the role of BRCA1 in the other two DSB repair pathways, classical non-homologous end-joining (C-NHEJ) and alternative NHEJ (A-NHEJ), remains unclear...
August 2016: Journal of Radiation Research
Summer B Thyme, Alexander F Schier
Error-prone repair of DNA double-strand breaks (DSBs) has been postulated to occur through classical non-homologous end joining (NHEJ) in systems ranging from nematode somatic tissues to zebrafish embryos. Contrary to this model, we show that zebrafish embryos mutant for DNA polymerase theta (Polq), a critical component of alternative end joining (alt-EJ), cannot repair DSBs induced by CRISPR/Cas9 or ionizing radiation. In the absence of DSBs, polq mutants are phenotypically normal, but they do not survive mutagenesis and display dramatic differences in the mutation profiles compared with the wild-type...
April 13, 2016: Cell Reports
Sara Ahrabi, Sovan Sarkar, Sophia X Pfister, Giacomo Pirovano, Geoff S Higgins, Andrew C G Porter, Timothy C Humphrey
DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells...
July 8, 2016: Nucleic Acids Research
Kerstin Felgentreff, Sachin N Baxi, Yu Nee Lee, Kerry Dobbs, Lauren A Henderson, Krisztian Csomos, Erdyni N Tsitsikov, Mary Armanios, Jolan E Walter, Luigi D Notarangelo
PURPOSE: DNA Ligase 4 (LIG4) is a key factor in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway needed for V(D)J recombination and the generation of the T cell receptor and immunoglobulin molecules. Defects in LIG4 result in a variable syndrome of growth retardation, pancytopenia, combined immunodeficiency, cellular radiosensitivity, and developmental delay. METHODS: We diagnosed a patient with LIG4 syndrome by radiosensitivity testing on peripheral blood cells, and established that two of her four healthy siblings carried the same compound heterozygous LIG4 mutations...
May 2016: Journal of Clinical Immunology
Ganesan Govindan, Sivaprakash Ramalingam
Recent advances in the targeted genome engineering enable molecular biologists to generate sequence specific modifications with greater efficiency and higher specificity in complex eukaryotic genomes. Programmable site-specific DNA cleavage reagents and cellular DNA repair mechanisms have made this possible. These reagents have become powerful tools for delivering a site-specific genomic double-strand break (DSB) at the desired chromosomal locus, which produces sequence alterations through error-prone non-homologous end joining (NHEJ) resulting in gene inactivations/knockouts...
November 2016: Journal of Cellular Physiology
Kate Liddiard, Brian Ruis, Taylor Takasugi, Adam Harvey, Kevin E Ashelford, Eric A Hendrickson, Duncan M Baird
Telomeres shorten with each cell division and can ultimately become substrates for nonhomologous end-joining repair, leading to large-scale genomic rearrangements of the kind frequently observed in human cancers. We have characterized more than 1400 telomere fusion events at the single-molecule level, using a combination of high-throughput sequence analysis together with experimentally induced telomeric double-stranded DNA breaks. We show that a single chromosomal dysfunctional telomere can fuse with diverse nontelomeric genomic loci, even in the presence of an otherwise stable genome, and that fusion predominates in coding regions...
May 2016: Genome Research
Bjoern Schwer, Pei-Chi Wei, Amelia N Chang, Jennifer Kao, Zhou Du, Robin M Meyers, Frederick W Alt
High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs...
February 23, 2016: Proceedings of the National Academy of Sciences of the United States of America
Guangqing Lu, Jinzhi Duan, Sheng Shu, Xuxiang Wang, Linlin Gao, Jing Guo, Yu Zhang
In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated...
February 2, 2016: Proceedings of the National Academy of Sciences of the United States of America
Shahnaz Masani, Li Han, Katheryn Meek, Kefei Yu
Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway...
February 2, 2016: Proceedings of the National Academy of Sciences of the United States of America
Emad A Ahmed, Harry Scherthan, Dirk G de Rooij
Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation...
2015: International Journal of Molecular Sciences
Li Zhu, Hiroaki Mon, Jian Xu, Jae Man Lee, Takahiro Kusakabe
Gene targeting can be achieved by precise genetic modifications through homology-directed repair (HDR) after DNA breaks introduced by genome editing tools such as CRISPR/Cas9 system. The most common form of HDR is homologous recombination (HR). Binding to the DNA breaks by HR factors is thought to compete with non-homologous end joining (NHEJ), an alternative DNA repair pathway. Here, we knocked out the factors in NHEJ by CRISPR/Cas9 system in silkworm cells, so that increased the activities of HR up to 7-fold...
2015: Scientific Reports
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