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non-homologous end joining

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https://www.readbyqxmd.com/read/29770864/agrobacterium-mediated-transformation-of-yeast-and-fungi
#1
Paul J J Hooykaas, G Paul H van Heusden, Xiaolei Niu, M Reza Roushan, Jalal Soltani, Xiaorong Zhang, Bert J van der Zaal
Two decades ago, it was discovered that the well-known plant vector Agrobacterium tumefaciens can also transform yeasts and fungi when these microorganisms are co-cultivated on a solid substrate in the presence of a phenolic inducer such as acetosyringone. It is important that the medium has a low pH (5-6) and that the temperature is kept at room temperature (20-25 °C) during co-cultivation. Nowadays, Agrobacterium-mediated transformation (AMT) is the method of choice for the transformation of many fungal species; as the method is simple, the transformation efficiencies are much higher than with other methods, and AMT leads to single-copy integration much more frequently than do other methods...
May 17, 2018: Current Topics in Microbiology and Immunology
https://www.readbyqxmd.com/read/29770349/single-step-precision-genome-editing-in-yeast-using-crispr-cas9
#2
Azat Akhmetov, Jon M Laurent, Jimmy Gollihar, Elizabeth C Gardner, Riddhiman K Garge, Andrew D Ellington, Aashiq H Kachroo, Edward M Marcotte
Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29769340/the-htlv-1-basic-leucine-zipper-factor-hbz-attenuates-repair-of-double-stranded-dna-breaks-via-non-homologous-end-joining-nhej
#3
A W Rushing, K Hoang, N Polakowski, I Lemasson
Adult T-cell leukemia (ATL) is a fatal malignancy of CD4+ T-cells infected with human T-cell leukemia virus type I (HTLV-1). ATL cells often exhibit random gross chromosomal rearrangements that are associated with the induction and improper repair of double-stranded DNA breaks (DSBs). The viral oncoprotein Tax has been reported to impair DSB repair, but is not shown to be consistently expressed throughout all phases of infection. The viral oncoprotein HTLV-1 basic leucine zipper factor (HBZ) is consistently expressed prior to and throughout disease progression, but it is unclear whether it also influences DSB repair...
May 16, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29760046/dual-hdac-and-pi3k-inhibition-abrogates-nf%C3%AE%C2%BAb-and-foxm1-mediated-dna-damage-response-to-radiosensitize-pediatric-high-grade-gliomas
#4
Sharmistha Pal, David Kozono, Xiaodong Yang, Wojciech Fendler, Whitney Fitts, Jing Ni, John A Alberta, Jean Zhao, Kevin X Liu, Jie Bian, Nathalene Truffaux, William A Weiss, Adam C Resnick, Pratiti Bandopadhayay, Keith L Ligon, Steven G Dubois, Sabine Mueller, Dipanjan Chowdhury, Daphne A Haas-Kogan
Aberrant chromatin remodeling and activation of the phosphatidylinositol 3-kinase (PI3K) pathway have been identified as important mediators of pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) pathogenesis. As inhibition of these pathways are promising therapeutic avenues and radiation is the only modality to prolong survival of DIPG patients, we sought to explore radiosensitizing functions of such inhibition and explore mechanisms of action of such agents. Here we demonstrate that combined treatment with radiotherapy and CUDC-907, a novel first-in-class dual inhibitor of histone deacetylases (HDAC) and PI3K, evokes a potent cytotoxic response in pHGG and DIPG models...
May 14, 2018: Cancer Research
https://www.readbyqxmd.com/read/29754232/targeted-gene-knock-out-using-nuclease-assisted-vector-integration-hemi-and-homozygous-deletion-of-jag1
#5
Michael Gapinske, Nathan Tague, Jackson Winter, Gregory H Underhill, Pablo Perez-Pinera
Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29749794/molecular-cytogenetics-guides-massively-parallel-sequencing-of-a-radiation-induced-chromosome-translocation-in-human-cells
#6
Michael N Cornforth, Pavana Anur, Nicholas Wang, Erin Robinson, F Andrew Ray, Joel S Bedford, Bradford D Loucas, Eli S Williams, Myron Peto, Paul Spellman, Rahul Kollipara, Ralf Kittler, Joe W Gray, Susan M Bailey
Chromosome rearrangements are large-scale structural variants that are recognized drivers of oncogenic events in cancers of all types. Cytogenetics allows for their rapid, genome-wide detection, but does not provide gene-level resolution. Massively parallel sequencing (MPS) promises DNA sequence-level characterization of the specific breakpoints involved, but is strongly influenced by bioinformatics filters that affect detection efficiency. We sought to characterize the breakpoint junctions of chromosomal translocations and inversions in the clonal derivatives of human cells exposed to ionizing radiation...
May 11, 2018: Radiation Research
https://www.readbyqxmd.com/read/29739874/pten-regulates-non-homologous-end-joining-by-epigenetic-induction-of-nhej1-xlf
#7
Parker L Sulkowski, Susan E Scanlon, Sebastian Oeck, Peter M Glazer
DNA double-strand breaks (DSBs) are the most cytotoxic DNA lesions, and up to 90% of DSBs require repair by non-homologous end joining (NHEJ). Functional and genomic analyses of patient-derived melanomas revealed that PTEN loss is associated with NHEJ deficiency. In PTEN-null melanomas PTEN complementation rescued the NHEJ defect; conversely suppression of PTEN compromised NHEJ. Mechanistic studies revealed that PTEN promotes NHEJ through direct induction of expression of XRCC4-like factor (NHEJ1/XLF) which functions in DNA end bridging and ligation...
May 8, 2018: Molecular Cancer Research: MCR
https://www.readbyqxmd.com/read/29734518/zinc-finger-nuclease-zfn-mediated-precision-genome-editing-of-an-endogenous-gene-in-hexaploid-bread-wheat-triticum-aestivum-using-a-dna-repair-template
#8
Yidong Ran, Nicola Patron, Pippa Kay, Debbie Wong, Margaret Buchanan, Ying-Ying Cao, Tim Sawbridge, John P Davies, John Mason, Steven R Webb, German Spangenberg, W Michael Ainley, Terence A Walsh, Matthew J Hayden
Sequence-specific nucleases have been used to engineer targeted genome modifications in various plants. While targeted gene knockouts resulting in loss-of-function have been reported with relatively high rates of success, targeted gene editing using an exogenously supplied DNA repair template and site-specific transgene integration has been more challenging. Here, we report the first application of Zinc Finger Nuclease (ZFN)-mediated, non-homologous end joining (NHEJ)-directed editing of a native gene in allohexaploid bread wheat to introduce, via a supplied DNA repair template, a specific single amino acid change into the coding sequence of acetohydroxyacid synthase (AHAS) to confer resistance to imidazolinone herbicides...
May 7, 2018: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/29721206/stress-and-stem-cells-adult-muse-cells-tolerate-extensive-genotoxic-stimuli-better-than-mesenchymal-stromal-cells
#9
Nicola Alessio, Tiziana Squillaro, Servet Özcan, Giovanni Di Bernardo, Massimo Venditti, Mariarosa Melone, Gianfranco Peluso, Umberto Galderisi
Mesenchymal stromal cells (MSCs) are not a homogenous population but comprehend several cell types, such as stem cells, progenitor cells, fibroblasts, and other types of cells. Among these is a population of pluripotent stem cells, which represent around 1-3% of MSCs. These cells, named multilineage-differentiating stress enduring (Muse) cells, are stress-tolerant cells. Stem cells may undergo several rounds of intrinsic and extrinsic stresses due to their long life and must have a robust and effective DNA damage checkpoint and DNA repair mechanism, which, following a genotoxic episode, promote the complete recovery of cells rather than triggering senescence and/or apoptosis...
April 10, 2018: Oncotarget
https://www.readbyqxmd.com/read/29710373/transforming-plant-biology-and-breeding-with-crispr-cas9-cas12-and-cas13
#10
REVIEW
Patrick Schindele, Felix Wolter, Holger Puchta
Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone non-homologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change of plant genomes via homologous recombination have recently been developed...
April 30, 2018: FEBS Letters
https://www.readbyqxmd.com/read/29698738/uranyl-acetate-induced-dna-single-strand-breaks-and-ap-sites-in-chinese-hamster-ovary-cells
#11
Monica Yellowhair, Michelle R Romanotto, Diane M Stearns, R Clark Lantz
The aim of this study is to characterize the genotoxicity of depleted uranium (DU) in Chinese Hamster Ovary cells (CHO) with mutations in various DNA repair pathways. CHO cells were exposed to 0-300 μM of soluble DU as uranyl acetate (UA) for 0-48 h. Intracellular UA concentrations were measured via inductively coupled mass spectrometry (ICP-MS) and visualized by transmission electron microscopy (TEM). Cytotoxicity was assessed in vitro by clonogenic survival assay. DNA damage response was assessed via Fast Micromethod® to determine UA-induced DNA single strand breaks...
April 24, 2018: Toxicology and Applied Pharmacology
https://www.readbyqxmd.com/read/29685705/genistein-sensitizes-glioblastoma-cells-to-carbon-ions-via-inhibiting-dna-pkcs-phosphorylation-and-subsequently-repressing-nhej-and-delaying-hr-repair-pathways
#12
Xiongxiong Liu, Ping Li, Ryoichi Hirayama, Yuzhen Niu, Xinguo Liu, Weiqiang Chen, Xiaodong Jin, Pengcheng Zhang, Fei Ye, Ting Zhao, Bingtao Liu, Qiang Li
BACKGROUND AND PURPOSE: Previously, we found genistein could sensitize cancer cells to low linear energy transfer (LET) X-rays via inhibiting DNA-PKcs activities. Especially, high-LET heavy ion produces more DNA double strand breaks (DSBs) than low-LET radiation. Thus, the study was designed to investigate the detailed molecular mechanisms of genistein on sensitizing cancer cells to heavy ions. MATERIALS AND METHODS: Human glioblastoma (GBM) cell lines with or without genistein pre-treatment were irradiated with high-LET carbon ions...
April 20, 2018: Radiotherapy and Oncology: Journal of the European Society for Therapeutic Radiology and Oncology
https://www.readbyqxmd.com/read/29676720/mutagenic-repair-of-double-stranded-dna-breaks-in-vaccinia-virus-genomes-requires-cellular-dna-ligase-iv-activity-in-the-cytosol
#13
Rutger David Luteijn, Ingo Drexler, Geoffrey L Smith, Robert Jan Lebbink, Emmanuel J H J Wiertz
Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes...
April 20, 2018: Journal of General Virology
https://www.readbyqxmd.com/read/29675030/homology-directed-repair-of-a-defective-glabrous-gene-in-arabidopsis-with-cas9-based-gene-targeting
#14
Florian Hahn, Marion Eisenhut, Otho Mantegazza, Andreas P M Weber
The CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell's own repair machinery either by the non-homologous end joining pathway or by homologous recombination (HR). While the first repair mechanism results in random mutations at the double-strand break site, HR uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome...
2018: Frontiers in Plant Science
https://www.readbyqxmd.com/read/29674014/quantitative-proteomics-of-bombyx-mori-after-bmnpv-challenge
#15
Fuxiang Mao, Jihai Lei, Obeng Enoch, Ming Wei, Cui Zhao, Yanping Quan, Wei Yu
The domesticated silkworm is an ideal and economic insect model that plays crucial roles in sericulture and bioreactor. Bombyx mori nucleopolyhedrovirus (BmNPV) is not only an infectious pathogen to B. mori, but also an efficient vector expressing recombinant proteins. Although, the proteomics of silkworm and BmN cell membrane lipid raft towards BmNPV infection had been investigated, proteome results of BmN cells upon BmNPV challenge currently remain ambiguous. In order to explore the interaction between silkworm and BmNPV, we analyzed several pivotal processes of BmNPV infected BmN cell by quantitative mass spectrometry...
April 16, 2018: Journal of Proteomics
https://www.readbyqxmd.com/read/29672770/clustered-regularly-interspaced-short-palindromic-repeats-crispr-crispr-associated-protein-9-with-improved-proof-reading-enhances-homology-directed-repair
#16
Tomoko Kato-Inui, Gou Takahashi, Szuyin Hsu, Yuichiro Miyaoka
Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) predominantly induces non-homologous end joining (NHEJ), which generates random insertions or deletions, whereas homology-directed repair (HDR), which generates precise recombination products, is useful for wider applications. However, the factors that determine the ratio of HDR to NHEJ products after CRISPR/Cas9 editing remain unclear, and methods by which the proportion of HDR products can be increased have not yet been fully established...
April 17, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29666389/attenuated-dna-damage-responses-and-increased-apoptosis-characterize-human-hematopoietic-stem-cells-exposed-to-irradiation
#17
Shahar Biechonski, Leonid Olender, Adi Zipin-Roitman, Muhammad Yassin, Nasma Aqaqe, Victoria Marcu-Malina, Melanie Rall-Scharpf, Magan Trottier, M Stephen Meyn, Lisa Wiesmüller, Katia Beider, Yael Raz, Dan Grisaru, Arnon Nagler, Michael Milyavsky
Failure to precisely repair DNA damage in self-renewing Hematopoietic Stem and early Progenitor Cells (HSPCs) can disrupt normal hematopoiesis and promote leukemogenesis. Although HSPCs are widely considered a target of ionizing radiation (IR)-induced hematopoietic injury, definitive data regarding cell death, DNA repair, and genomic stability in these rare quiescent cells are scarce. We found that irradiated HSPCs, but not lineage-committed progenitors (CPs), undergo rapid ATM-dependent apoptosis, which is suppressed upon interaction with bone-marrow stroma cells...
April 17, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29658569/%C3%AE-%C3%A2-catenin-nuclear-translocation-induced-by-hif%C3%A2-1%C3%AE-overexpression-leads-to-the-radioresistance-of-prostate-cancer
#18
Yong Luo, Mingchuan Li, Xuemei Zuo, Spyridon P Basourakos, Jiao Zhang, Jiahui Zhao, Yili Han, Yunhua Lin, Yongxing Wang, Yongguang Jiang, Ling Lan
Hypoxia-inducible factor‑1α (HIF‑1α) is known to play crucial roles in tumor radioresistance; however, the molecular mechanisms responsible for the promotion of tumor radioresistance by HIF‑1α remain unclear. β‑catenin is known to be involved in the metastatic potential of prostate cancer (PCa). In this study, to investigate the role of HIF‑1α and β‑catenin in the radioresistance of PCa, two PCa cell lines, LNCaP and C4‑2B, were grouped as follows: Negative control (no treatment), HIF‑1α overexpression group (transfected with HIF‑1α overexpression plasmid) and β‑catenin silenced group (transfected with HIF‑1α plasmids and β‑catenin-shRNA)...
April 12, 2018: International Journal of Oncology
https://www.readbyqxmd.com/read/29625543/mechanisms-of-mitochondrial-dna-repair-in-mammals
#19
REVIEW
L A Zinovkina
Accumulation of mutations in mitochondrial DNA leads to the development of severe, currently untreatable diseases. The contribution of these mutations to aging and progress of neurodegenerative diseases is actively studied. Elucidation of DNA repair mechanisms in mitochondria is necessary for both developing approaches to the therapy of diseases caused by mitochondrial mutations and understanding specific features of mitochondrial genome functioning. Mitochondrial DNA repair systems have become a subject of extensive studies only in the last decade due to development of molecular biology methods...
March 2018: Biochemistry. Biokhimii︠a︡
https://www.readbyqxmd.com/read/29622782/multiple-sgrnas-with-overlapping-sequences-enhance-crispr-cas9-mediated-knock-in-efficiency
#20
Da Eun Jang, Jae Young Lee, Jae Hoon Lee, Ok Jae Koo, Hee Sook Bae, Min Hee Jung, Ji Hyun Bae, Woo Sung Hwang, Yoo Jin Chang, Yoon Hoo Lee, Han Woong Lee, Su Cheong Yeom
The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation...
April 6, 2018: Experimental & Molecular Medicine
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