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https://www.readbyqxmd.com/read/29180609/honokiol-radiosensitizes-squamous-cell-carcinoma-of-the-head-and-neck-by-downregulation-of-survivin
#1
Xu Wang, Jonathan J Beitler, Wen Huang, Guo Chen, Guoqing Qian, Kelly R Magliocca, Mihir R Patel, Amy Y Chen, Jun Zhang, Sreenivas Nannapaneni, Sungjin Kim, Zhengjia Chen, Xingming Deng, Nabil F Saba, Zhuo Georgia Chen, Jack Arbiser, Dong M Shin
PURPOSE: Previous studies revealed diverging results regarding the role of survivin in squamous cell carcinoma of the head and neck (SCCHN).This study aimed to evaluate the clinical significance of survivin expression in SCCHN; the function of survivin in DNA damage repair following ionizing radiationtherapy (RT) in SCCHN cells; and the potential of honokiol to enhance RT through downregulation of survivin. EXPERIMENTAL DESIGN: Expression of survivin in SCCHN patient primary tumor tissues (n=100) was analyzed and correlated with clinical parameters...
November 27, 2017: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
https://www.readbyqxmd.com/read/29155472/experimental-reconstruction-of-double-strand-break-repair-mediated-plastid-dna-insertion-into-the-tobacco-nucleus
#2
Dong Wang, Jinbao Gu, Rakesh David, Zhen Wang, Songtao Yang, Iain R Searle, Jian-Kang Zhu, Jeremy N Timmis
The mitochondria and plastids of eukaryotic cells evolved from endosymbiotic prokaryotes. DNA from the endosymbionts has bombarded nuclei since the ancestral prokaryotes were engulfed by a precursor of the nucleated eukaryotic host. An experimental confirmation regarding the molecular mechanisms responsible for organelle DNA incorporation into nuclei has not been performed until the present analysis. Here we introduced double-strand DNA breaks into the nuclear genome of tobacco through inducible expression of I-SceI, and showed experimentally that tobacco chloroplast DNAs insert into nuclear genomes through double-strand DNA break repair...
November 20, 2017: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/29121353/secondary-structure-forming-sequences-drive-sd-mmej-repair-of-dna-double-strand-breaks
#3
Varandt Y Khodaverdian, Terrence Hanscom, Amy Marie Yu, Taylor L Yu, Victoria Mak, Alexander J Brown, Steven A Roberts, Mitch McVey
Alternative end-joining (alt-EJ) repair of DNA double-strand breaks is associated with deletions, chromosome translocations, and genome instability. Alt-EJ frequently uses annealing of microhomologous sequences to tether broken ends. When accessible pre-existing microhomologies do not exist, we have postulated that new microhomologies can be created via limited DNA synthesis at secondary-structure forming sequences. This model, called synthesis-dependent microhomology-mediated end joining (SD-MMEJ), predicts that differences between DNA sequences near double-strand breaks should alter repair outcomes in predictable ways...
November 7, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29083855/repair-of-a-site-specific-dna-cleavage-old-school-lessons-for-cas9-mediated-gene-editing
#4
Danielle N Gallagher, James E Haber
CRISPR/Cas9-mediated gene editing may involve nonhomologous end-joining to create various insertion/deletions (indels) or may employ homologous recombination to modify precisely the target DNA sequence. Our understanding of these processes has been guided by earlier studies using other site-specific endonucleases, both in model organisms such as budding yeast and in mammalian cells. We briefly review what has been gleaned from such studies using the HO and I-SceI endonucleases and how these findings guide current gene editing strategies...
November 14, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/29078113/tdp1-is-required-for-efficient-non-homologous-end-joining-in-human-cells
#5
Jing Li, Matthew Summerlin, Karin C Nitiss, John L Nitiss, Leslyn A Hanakahi
Tyrosyl-DNA phosphodiesterase 1 (TDP1) can remove a wide variety of 3' and 5' terminal DNA adducts. Genetic studies in yeast identified TDP1 as a regulator of non-homologous end joining (NHEJ) fidelity in the repair of double-strand breaks (DSBs) lacking terminal adducts. In this communication, we show that TDP1 plays an important role in joining cohesive DSBs in human cells. To investigate the role of TDP1 in NHEJ in live human cells we used CRISPR/cas9 to produce TDP1-knockout (TDP1-KO) HEK-293 cells. As expected, human TDP1-KO cells were highly sensitive to topoisomerase poisons and ionizing radiation...
December 2017: DNA Repair
https://www.readbyqxmd.com/read/29065183/an-electroporation-free-method-based-on-red-recombineering-for-markerless-deletion-and-genomic-replacement-in-the-escherichia-coli-dh1-genome
#6
Yanlong Wei, Pingping Deng, Ali Mohsin, Yan Yang, Huayan Zhou, Meijin Guo, Hongqing Fang
The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach...
2017: PloS One
https://www.readbyqxmd.com/read/29055804/high-homology-is-not-required-at-the-site-of-strand-invasion-during-recombinational-double-strand-break-repair-in-mammalian-chromosomes
#7
Kristina M Chapman, Megan M Wilkey, Kendall E Potter, Barbara C Waldman, Alan S Waldman
We investigated the impact of sequence divergence on DNA double-strand break (DSB) repair occurring via recombination in cultured thymidine kinase deficient mouse fibroblasts. We stably transfected cells with a DNA construct harboring a herpes thymidine kinase (tk) gene (the "recipient") rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I-SceI. The construct also contained a closely linked truncated "donor" tk sequence. The donor could potentially restore function to the recipient gene via recombination provoked by induction of a DSB at the I-SceI site in the recipient...
December 2017: DNA Repair
https://www.readbyqxmd.com/read/28973861/ctcf-prevents-genomic-instability-by-promoting-homologous-recombination-directed-dna-double-strand-break-repair
#8
Fengchao Lang, Xin Li, Wenhai Zheng, Zhuoran Li, Danfeng Lu, Guijun Chen, Daohua Gong, Liping Yang, Jinlin Fu, Peng Shi, Jumin Zhou
CTCF is an essential epigenetic regulator mediating chromatin insulation, long-range regulatory interactions, and the organization of large topological domains in the nucleus. Phenotypes of CTCF haploinsufficient mutations in humans, knockout in mice, and depletion in cells are often consistent with impaired genome stability, but a role of CTCF in genome maintenance has not been fully investigated. Here, we report that CTCF maintains genome stability, is recruited to sites of DNA damage, and promotes homologous recombination repair of DNA double-strand breaks (DSBs)...
October 10, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28911102/fancj-helicase-controls-the-balance-between-short-and-long-tract-gene-conversions-between-sister-chromatids
#9
Sarmi Nath, Kumar Somyajit, Anup Mishra, Ralph Scully, Ganesh Nagaraju
The FANCJ DNA helicase is linked to hereditary breast and ovarian cancers as well as bone marrow failure disorder Fanconi anemia (FA). Although FANCJ has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), the molecular mechanism underlying the tumor suppressor functions of FANCJ remains obscure. Here, we demonstrate that FANCJ deficient human and hamster cells exhibit reduction in the overall gene conversions in response to a site-specific chromosomal DSB induced by I-SceI endonuclease...
September 6, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28911000/dicer-regulates-non-homologous-end-joining-and-is-associated-with-chemosensitivity-in-colon-cancer-patients
#10
Xiao Chen, Wen-Feng Li, Xiaoli Wu, Heng-Chao Zhang, Li Chen, Pei-Ying Zhang, Li-Yuan Liu, Di Ma, Tongke Chen, Lingli Zhou, Yunsheng Xu, Meng-Tao Zhou, Kai-Fu Tang
DNA double-strand break (DSB) repair is an important mechanism underlying chemotherapy resistance in human cancers. Dicer participates in DSB repair by facilitating homologous recombination. However, whether Dicer is involved in non-homologous end joining (NHEJ) remains unknown. Here, we addressed whether Dicer regulates NHEJ and chemosensitivity in colon cancer cells. Using our recently developed NHEJ assay, we found that DSB introduction by I-SceI cleavage leads to Dicer upregulation. Dicer knockdown increased SIRT7 binding and decreased the level of H3K18Ac (acetylated lysine 18 of histone H3) at DSB sites, thereby repressing the recruitment of NHEJ factors to DSB sites and inhibiting NHEJ...
September 1, 2017: Carcinogenesis
https://www.readbyqxmd.com/read/28878819/construction-of-an-easy-to-use-crispr-cas9-system-by-patching-a-newly-designed-exit-circuit
#11
Qiang Tang, Chunbo Lou, Shuang-Jiang Liu
BACKGROUND: Plasmid-borne genetic editing tools, including the widely used CRISPR-Cas9 system, have greatly facilitated bacterial programming to obtain novel functionalities. However, the lack of effective post-editing plasmid elimination methods impedes follow-up genetic manipulation or application. Conventional strategies including exposure to physical and chemical treatments, or exploiting temperature-sensitive replication origins have several drawbacks (e.g., they are limited for efficiency and are time-consuming)...
2017: Journal of Biological Engineering
https://www.readbyqxmd.com/read/28817121/meganuclease-assisted-generation-of-stable-transgenics-in-the-sea-anemone-nematostella-vectensis
#12
Eduard Renfer, Ulrich Technau
The sea anemone Nematostella vectensis is a model system used by a rapidly growing research community for comparative genomics, developmental biology and ecology. Here, we describe a microinjection procedure for creating stable transgenic lines in Nematostella based on meganuclease (I-SceI)-assisted integration of a transgenic cassette into the genome. The procedure describes the preparation of the reagents, microinjection of the transgenesis vector and the husbandry of transgenic animals. The microinjection setup differs from those of previously published protocols by the use of a holding capillary mounted on an inverted fluorescence microscope...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28783349/orthogonal-ribosome-bio-firewall
#13
Bin Jia, Hao Qi, Bing-Zhi Li, Shuo Pan, Duo Liu, Hong Liu, Yizhi Cai, Ying-Jin Yuan
Biocontainment systems are crucial for preventing genetically modified organisms from escaping into natural ecosystems. Here, we describe the orthogonal ribosome bio-firewall, which consists of an activation circuit and a degradation circuit. The activation circuit is a genetic AND gate based on activation of the encrypted pathway by the orthogonal ribosome in response to specific environmental signals. The degradation circuit is a genetic NOT gate with an output of I-SceI homing endonuclease, which conditionally degrades the orthogonal ribosome genes...
August 7, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28582396/participation-of-androgen-and-its-receptor-in-sex-determination-of-an-amphibian-species
#14
Akira Oike, Maho Kodama, Shigeki Yasumasu, Takashi Yamamoto, Yoriko Nakamura, Etsuro Ito, Masahisa Nakamura
INTRODUCTION: In the Japanese frog Rana (R.) rugosa the androgen receptor (AR) gene on the W chromosome (W-AR) is barely expressed. Previously we showed that incomplete female-to-male sex-reversal occurred in Z-AR transgenic female frogs. To date, however, there is no report showing that AR with androgens can determine genetically programed male sex fate in any vertebrate species. Here, we examined whether AR together with androgens functions as a sex determinant in an amphibian species...
2017: PloS One
https://www.readbyqxmd.com/read/28336775/nelf-e-is-recruited-to-dna-double-strand-break-sites-to-promote-transcriptional-repression-and-repair
#15
Samah W Awwad, Enas R Abu-Zhayia, Noga Guttmann-Raviv, Nabieh Ayoub
Double-strand breaks (DSBs) trigger rapid and transient transcription pause to prevent collisions between repair and transcription machineries at damage sites. Little is known about the mechanisms that ensure transcriptional block after DNA damage. Here, we reveal a novel role of the negative elongation factor NELF in blocking transcription activity nearby DSBs. We show that NELF-E and NELF-A are rapidly recruited to DSB sites. Furthermore, NELF-E recruitment and its repressive activity are both required for switching off transcription at DSBs...
May 2017: EMBO Reports
https://www.readbyqxmd.com/read/28212413/scarless-deletion-of-up-to-seven-methyl-accepting-chemotaxis-genes-with-an-optimized-method-highlights-key-function-of-chem-in-salmonella-typhimurium
#16
Stefanie Hoffmann, Christiane Schmidt, Steffi Walter, Jennifer K Bender, Roman G Gerlach
Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus...
2017: PloS One
https://www.readbyqxmd.com/read/28000382/aging-impairs-double-strand-break-repair-by-homologous-recombination-in-drosophila-germ-cells
#17
Laetitia Delabaere, Henry A Ertl, Dashiell J Massey, Carolyn M Hofley, Faraz Sohail, Elisa J Bienenstock, Hans Sebastian, Irene Chiolo, Jeannine R LaRocque
Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR)...
April 2017: Aging Cell
https://www.readbyqxmd.com/read/27924006/the-homologous-recombination-protein-rad51d-protects-the-genome-from-large-deletions
#18
Wade A Reh, Rodney S Nairn, Megan P Lowery, Karen M Vasquez
Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that protects the genome from chromosomal instability. RAD51 mediator proteins (i.e. paralogs) are critical for efficient HR in mammalian cells. However, how HR-deficient cells process DSBs is not clear. Here, we utilized a loss-of-function HR-reporter substrate to simultaneously monitor HR-mediated gene conversion and non-conservative mutation events. The assay is designed around a heteroallelic duplication of the Aprt gene at its endogenous locus in isogenic Chinese hamster ovary cell lines...
February 28, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/27825743/quantification-and-genome-wide-mapping-of-dna-double-strand-breaks
#19
Marie-Chantal Grégoire, Julien Massonneau, Frédéric Leduc, Mélina Arguin, Marc-André Brazeau, Guylain Boissonneault
DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase...
December 2016: DNA Repair
https://www.readbyqxmd.com/read/27798638/53bp1-protects-against-ctip-dependent-capture-of-ectopic-chromosomal-sequences-at-the-junction-of-distant-double-strand-breaks
#20
Josée Guirouilh-Barbat, Camille Gelot, Anyong Xie, Elodie Dardillac, Ralph Scully, Bernard S Lopez
DNA double-strand breaks (DSB) are very harmful lesions that can generate genome rearrangements. In this study, we used intrachromosomal reporters to compare both the efficiency and accuracy of end-joining occurring with close (34 bp apart) vs. distant DSBs (3200 bp apart) in human fibroblasts. We showed that a few kb between two intrachromosomal I-SceI-induced DSBs are sufficient to foster deletions and capture/insertions at the junction scar. Captured sequences are mostly coupled to deletions and can be partial duplications of the reporter (i...
October 2016: PLoS Genetics
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