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Homologous recombination

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https://www.readbyqxmd.com/read/28723575/crispr-mediated-integration-of-large-gene-cassettes-using-aav-donor-vectors
#1
Rasmus O Bak, Matthew H Porteus
The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno-associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of ∼4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing...
July 18, 2017: Cell Reports
https://www.readbyqxmd.com/read/28722656/corrupting-the-dna-damage-response-a-critical-role-for-rad52-in-tumor-cell-survival
#2
Rachel Lieberman, Ming You
The DNA damage response enables cells to survive, maintain genome integrity, and to safeguard the transmission of high-fidelity genetic information. Upon sensing DNA damage, cells respond by activating this multi-faceted DNA damage response leading to restoration of the cell, senescence, programmed cell death, or genomic instability if the cell survives without proper repair. However, unlike normal cells, cancer cells maintain a marked level of genomic instability. Because of this enhanced propensity to accumulate DNA damage, tumor cells rely on homologous recombination repair as a means of protection from the lethal effect of both spontaneous and therapy-induced double-strand breaks (DSBs) in DNA...
July 15, 2017: Aging
https://www.readbyqxmd.com/read/28721587/genetically-engineered-cell-lines-for-%C3%AE-1-antitrypsin-expression
#3
Qianqian Ji, Caiping Guo, Chen Xie, Yingdan Wu, Pei Zhang, Hui Li, Yongjun Lu
OBJECTIVES: To establish genetically modified cell lines that can produce functional α1-antitrypsin (AAT), by CRISPR/Cas9-assisted homologous recombination. RESULTS: α1-Antitrypsin deficiency (AATD) is a monogenic heritable disease that often results in lungs and liver damage. Current augmentation therapy is expensive and in short of supply. To develop a safer and more effective therapeutic strategy for AATD, we integrated the AAT gene (SERPINA1, NG_008290.1) into the AAVS1 locus of human cell line HEK293T and assessed the safety and efficacy of CRISPR/Cas9 on producing potential therapeutic cell lines...
July 18, 2017: Biotechnology Letters
https://www.readbyqxmd.com/read/28719308/a-recombinant-47-kda-outer-membrane-protein-induces-an-immune-response-against-orientia-tsutsugamushi-strain-boryong
#4
Sangho Choi, Hang Jin Jeong, Kyu-Jam Hwang, Byoungchul Gill, Young Ran Ju, Yeong Seon Lee, Jeongmin Lee
We investigated the 47-kDa outer membrane protein (OMP), which is a periplasmic serine protease and an antigenic major surface protein of Orientia tsutsugamushi, as a vaccine candidate. We developed a conventional subunit vaccine expressing recombinant 47-kDa OMP (rec47) and a DNA vaccine (p47). In mouse immunization experiments, intranasal immunization with rec47 alone or with rec47 plus heat-labile enterotoxin B subunit from Escherichia coli or plus cholera toxin (CT) as adjuvants induced a higher amount of rec47-specific antibodies than intramuscular immunization with p47 alone or with p47 plus pBOOST2-samIRF7/3 (pB) as adjuvant...
July 2017: American Journal of Tropical Medicine and Hygiene
https://www.readbyqxmd.com/read/28718810/dna2-an-important-player-in-dna-damage-response-or-just-another-dna-maintenance-protein
#5
REVIEW
Elzbieta Pawłowska, Joanna Szczepanska, Janusz Blasiak
The human DNA2 (DNA replication helicase/nuclease 2) protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER), interacting with the replication protein A (RPA) and the flap endonuclease 1 (FEN1). DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN) and Bloom syndrome protein (BLM)...
July 18, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28718632/systematic-approaches-to-efficiently-produce-2-3-butanediol-in-a-marine-cyanobacterium
#6
Nicole E Nozzi, Anna E Case, Austin L Carroll, Shota Atsumi
Cyanobacteria have attracted significant interest as a platform for renewable production of fuel and feedstock chemicals from abundant atmospheric carbon dioxide by way of photosynthesis. While great strides have been made in developing this technology in freshwater cyanobacteria, logistical issues remain in scale-up. Use of the cyanobacterium Synechococcus sp. PCC 7002 (7002) as a chemical production chassis could address a number of these issues given the higher tolerance to salt, light, and heat as well as the fast growth rate of 7002 in comparison to traditional model cyanobacteria such as Synechococcus elongatus PCC 7942 and Synechocystis sp...
July 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28718400/histone-h3g34r-mutation-causes-replication-stress-homologous-recombination-defects-and-genomic-instability-in-s-pombe
#7
Rajesh K Yadav, Carolyn M Jablonowski, Alfonso G Fernandez, Brandon R Lowe, Ryan A Henry, David Finkelstein, Kevin J Barnum, Alison L Pidoux, Yin-Ming Kuo, Jie Huang, Matthew J O'Connell, Andrew J Andrews, Arzu Onar-Thomas, Robin C Allshire, Janet F Partridge
Recurrent somatic mutations of H3F3A in aggressive pediatric high-grade gliomas generate K27M or G34R/V mutant histone H3.3. H3.3-G34R/V mutants are common in tumors with mutations in p53 and ATRX, an H3.3-specific chromatin remodeler. To gain insight into the role of H3-G34R, we generated fission yeast that express only the mutant histone H3. H3-G34R specifically reduces H3K36 tri-methylation and H3K36 acetylation, and mutants show partial transcriptional overlap with set2 deletions. H3-G34R mutants exhibit genomic instability and increased replication stress, including slowed replication fork restart, although DNA replication checkpoints are functional...
July 18, 2017: ELife
https://www.readbyqxmd.com/read/28718045/full-length-genome-sequence-analysis-of-four-subgroup-j-avian-leukosis-virus-strains-isolated-from-chickens-with-clinical-hemangioma
#8
Lulu Lin, Peikun Wang, Yongli Yang, Haijuan Li, Teng Huang, Ping Wei
Since 2014, cases of hemangioma associated with avian leukosis virus subgroup J (ALV-J) have been emerging in commercial chickens in Guangxi. In this study, four strains of the subgroup J avian leukosis virus (ALV-J), named GX14HG01, GX14HG04, GX14LT07, and GX14ZS14, were isolated from chickens with clinical hemangioma in 2014 by DF-1 cell culture and then identified with ELISA detection of ALV group specific antigen p27, the detection of subtype specific PCR and indirect immunofluorescence assay (IFA) with ALV-J specific monoclonal antibody...
July 17, 2017: Virus Genes
https://www.readbyqxmd.com/read/28717875/mast-like-protein-kinase-ireh1-from-arabidopsis-thaliana-co-localizes-with-the-centrosome-when-expressed-in-animal-cells
#9
Elena M Chudinova, Pavel A Karpov, Artem I Fokin, Alla I Yemets, Dmytro I Lytvyn, Elena S Nadezhdina, Yaroslav B Blume
The similarity of IREH1 (Incomplete Root Hair Elongation 1) and animal MAST kinases was confirmed; IREH1cDNA was cloned while expressing in cultured animal cells co-localized with the centrosome. In mammals and fruit flies, microtubule-associated serine/threonine-protein kinases (MAST) are strongly involved in the regulation of the microtubule system. Higher plants also possess protein kinases homologous to MASTs, but their function and interaction with the cytoskeleton remain unclear. Here, we confirmed the sequence and structural similarity of MAST-related putative protein kinase IREH1 (At3g17850) and known animal MAST kinases...
July 17, 2017: Planta
https://www.readbyqxmd.com/read/28716408/molecular-and-biochemical-characterization-of-a-vigna-mungo-map-kinase-associated-with-mungbean-yellow-mosaic-india-virus-infection-and-deciphering-its-role-in-restricting-the-virus-multiplication
#10
Anju Patel, Nrisingha Dey, Shubho Chaudhuri, Amita Pal
Yellow Mosaic Disease caused by the begomovirus Mungbean Yellow Mosaic India Virus (MYMIV) severely affects many economically important legumes. Recent investigations in Vigna mungo - MYMIV incompatible interaction identified a MAPK homolog in the defense signaling pathway. An important branch of immunity involves phosphorylation by evolutionary conserved Mitogen-activated protein kinases (MAPK) that transduce signals of pathogen invasion to downstream molecules leading to diverse immune responses. However, most of the knowledge of MAPKs is derived from model crops, and functions of these versatile kinases are little explored in legumes...
September 2017: Plant Science: An International Journal of Experimental Plant Biology
https://www.readbyqxmd.com/read/28716362/phosphoenolpyruvate-phosphotransferase-system-components-positively-regulate-klebsiella-biofilm-formation
#11
Yu-Tze Horng, Chi-Jen Wang, Wen-Ting Chung, Huei-Jen Chao, Yih-Yuan Chen, Po-Chi Soo
BACKGROUND/PURPOSE: Klebsiella pneumoniae is one of the leading causes of device-related infections (DRIs), which are associated with attachment of bacteria to these devices to form a biofilm. The latter is composed of not only bacteria but also extracellular polymeric substances (EPSes) consisting of extracellular DNAs, polysaccharides, and other macromolecules. The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) regulates diverse processes of bacterial physiology...
June 29, 2017: Journal of Microbiology, Immunology, and Infection, Wei Mian Yu Gan Ran za Zhi
https://www.readbyqxmd.com/read/28714850/high-resolution-mapping-of-heteroduplex-dna-formed-during-uv-induced-and-spontaneous-mitotic-recombination-events-in-yeast
#12
Yi Yin, Margaret Dominska, Eunice Yim, Thomas D Petes
In yeast, DNA breaks are usually repaired by homologous recombination (HR). An early step for HR pathways is formation of a heteroduplex, in which a single-strand from the broken DNA molecule pairs with a strand derived from an intact DNA molecule. If the two strands of DNA are not identical, there will be mismatches within the heteroduplex DNA (hetDNA). In wild-type strains, these mismatches are repaired by the mismatch repair (MMR) system, producing a gene conversion event. In strains lacking MMR, the mismatches persist...
July 17, 2017: ELife
https://www.readbyqxmd.com/read/28714471/progression-through-mitosis-promotes-parp-inhibitor-induced-cytotoxicity-in-homologous-recombination-deficient-cancer-cells
#13
Pepijn M Schoonen, Francien Talens, Colin Stok, Ewa Gogola, Anne Margriet Heijink, Peter Bouwman, Floris Foijer, Madalena Tarsounas, Sohvi Blatter, Jos Jonkers, Sven Rottenberg, Marcel A T M van Vugt
Mutations in homologous recombination (HR) genes BRCA1 and BRCA2 predispose to tumorigenesis. HR-deficient cancers are hypersensitive to Poly (ADP ribose)-polymerase (PARP) inhibitors, but can acquire resistance and relapse. Mechanistic understanding how PARP inhibition induces cytotoxicity in HR-deficient cancer cells is incomplete. Here we find PARP inhibition to compromise replication fork stability in HR-deficient cancer cells, leading to mitotic DNA damage and consequent chromatin bridges and lagging chromosomes in anaphase, frequently leading to cytokinesis failure, multinucleation and cell death...
July 17, 2017: Nature Communications
https://www.readbyqxmd.com/read/28714309/characterization-of-blebbistatin-inhibition-of-smooth-muscle-myosin-and-nonmuscle-myosin-2
#14
Hai-Man Zhang, Huan-Hong Ji, Tong Ni, Rong-Na Ma, Aibing Wang, Xiang-Dong Li
Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been determined and it remains controversial on the efficacy of blebbistatin inhibition of smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of the recombinant SmM and three NM2s (NM2a, 2b, and 2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50 values: 6...
July 17, 2017: Biochemistry
https://www.readbyqxmd.com/read/28713014/a-recombinant-type-2-porcine-reproductive-and-respiratory-syndrome-virus-between-nadc30-like-and-a-mlv-like-genetic-characterization-and-pathogenicity-for-piglets
#15
Ting Bian, Yingfeng Sun, Meng Hao, Lei Zhou, Xinna Ge, Xin Guo, Jun Han, Hanchun Yang
Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen for swine industry worldwide. The recombination occurring among PRRSV strains has been recognized as one of important molecular mechanisms for the evolution of PRRSV. Current prevalence of PRRSV NADC30-like causing clinical disease outbreaks is highly concerned in China. In the present study, the genetic characterization of a recombinant type 2 PRRSV (designated TJnh1501) was analyzed and its pathogenicity for piglets was examined...
July 13, 2017: Infection, Genetics and Evolution
https://www.readbyqxmd.com/read/28711786/exo1-suppresses-double-strand-break-induced-homologous-recombination-between-diverged-sequences-in-mammalian-cells
#16
Chun-Chin Chen, Elena Avdievich, Yongwei Zhang, Yu Zhang, Kaichun Wei, Kyeryoung Lee, Winfried Edelmann, Maria Jasin, Jeannine R LaRocque
DNA double-strand breaks (DSBs) can be repaired through several mechanisms, including homologous recombination (HR). While HR between identical sequences is robust in mammalian cells, HR between diverged sequences is suppressed by DNA mismatch-repair (MMR) components such as MSH2. Exonuclease I (EXO1) interacts with the MMR machinery and has been proposed to act downstream of the mismatch recognition proteins in mismatch correction. EXO1 has also been shown to participate in extensive DSB end resection, an initial step in the HR pathway...
July 10, 2017: DNA Repair
https://www.readbyqxmd.com/read/28710365/suppression-of-cell-migration-by-phospholipase-c-related-catalytically-inactive-protein-dependent-modulation-of-pi3k-signalling
#17
Satoshi Asano, Yuri Taniguchi, Yosuke Yamawaki, Jing Gao, Kae Harada, Hiroshi Takeuchi, Masato Hirata, Takashi Kanematsu
The metabolic processes of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] into PI(3,4,5)P3 and the subsequent PI(3,4,5)P3 signalling are involved in cell migration. Dysfunctions in the control of this pathway can cause human cancer cell migration and metastatic growth. Here we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a PI(4,5)P2-binding protein, regulates cancer cell migration. PRIP overexpression in MCF-7 and BT-549 human breast cancer cells inhibited cell migration in vitro and metastasis development in vivo...
July 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28710290/3-terminated-overhangs-regulate-dna-double-strand-break-processing-in-escherichia-coli
#18
Edyta Đermić, Davor Zahradka, Dušica Vujaklija, Siniša Ivanković, Damir Đermić
Double-strand breaks (DSBs) are lethal DNA lesions, which are repaired by homologous recombination in Escherichia coli To study DSB processing in vivo, we induced DSBs into the E. coli chromosome by gamma irradiation and measured chromosomal degradation. We show that the DNA degradation is regulated by RecA protein concentration and its rate of association with ssDNA. RecA decreased DNA degradation in wild-type, recB and recD strains, indicating that it is a general phenomenon in E. coli On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the recB1080 mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3' tail (ssExos)...
July 14, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28708824/tex19-1-promotes-spo11-dependent-meiotic-recombination-in-mouse-spermatocytes
#19
James H Crichton, Christopher J Playfoot, Marie MacLennan, David Read, Howard J Cooke, Ian R Adams
Meiosis relies on the SPO11 endonuclease to generate the recombinogenic DNA double strand breaks (DSBs) required for homologous chromosome synapsis and segregation. The number of meiotic DSBs needs to be sufficient to allow chromosomes to search for and find their homologs, but not excessive to the point of causing genome instability. Here we report that the mammal-specific gene Tex19.1 promotes Spo11-dependent recombination in mouse spermatocytes. We show that the chromosome asynapsis previously reported in Tex19...
July 14, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28708051/ralstonia-solanacearum-novel-e3-ubiquitin-ligase-nel-effectors-ripaw-and-ripar-suppress-pattern-triggered-immunity-in-plants
#20
Masahito Nakano, Kenji Oda, Takafumi Mukaihara
Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects more than 70 effector proteins into host plant cells via the Hrp type III secretion system to cause a successful infection. However, the function of these effectors in plant cells, especially in the suppression of plant immunity, remains largely unknown. In this study, we characterized two Ralstonia solanacearum effectors, RipAW and RipAR, which share homology with the IpaH family of effectors from animal and plant pathogenic bacteria, that have a novel E3 ubiquitin ligase (NEL) domain...
July 15, 2017: Microbiology
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