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microhomology mediated end join

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https://www.readbyqxmd.com/read/27894274/gene-cassette-knock-in-in-mammalian-cells-and-zygotes-by-enhanced-mmej
#1
Tomomi Aida, Shota Nakade, Tetsushi Sakuma, Yayoi Izu, Ayu Oishi, Keiji Mochida, Harumi Ishikubo, Takako Usami, Hidenori Aizawa, Takashi Yamamoto, Kohichi Tanaka
BACKGROUND: Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining. RESULTS: Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells...
November 28, 2016: BMC Genomics
https://www.readbyqxmd.com/read/27866150/crispr-cas9-induced-double-strand-break-repair-in-arabidopsis-non-homologous-end-joining-mutants
#2
Hexi Shen, Gary D Strunks, Bart J P M Klemann, Paul J J Hooykaas, Sylvia de Pater
Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs...
November 18, 2016: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/27849606/multiple-mechanisms-contribute-to-double-strand-break-repair-at-rereplication-forks-in-drosophila-follicle-cells
#3
Jessica L Alexander, Kelly Beagan, Terry L Orr-Weaver, Mitch McVey
Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. However, the primary mechanism used to repair rereplication DSBs varies across different experimental systems. In Drosophila follicle cells, developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. We have exploited this system to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency...
November 29, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27832076/complex-breakpoints-and-template-switching-associated-with-non-canonical-termination-of-homologous-recombination-in-mammalian-cells
#4
Andrea J Hartlerode, Nicholas A Willis, Anbazhagan Rajendran, John P Manis, Ralph Scully
A proportion of homologous recombination (HR) events in mammalian cells resolve by "long tract" gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i...
November 2016: PLoS Genetics
https://www.readbyqxmd.com/read/27798638/53bp1-protects-against-ctip-dependent-capture-of-ectopic-chromosomal-sequences-at-the-junction-of-distant-double-strand-breaks
#5
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
https://www.readbyqxmd.com/read/27623223/in%C3%A2-vivo-genome-editing-as-a-potential-treatment-strategy-for-inherited-retinal-dystrophies
#6
Mert Yanik, Brigitte Müller, Fei Song, Jacqueline Gall, Franziska Wagner, Wolfgang Wende, Birgit Lorenz, Knut Stieger
In vivo genome editing represents an emerging field in the treatment of monogenic disorders, as it may constitute a solution to the current hurdles in classic gene addition therapy, which are the low levels and limited duration of transgene expression. Following the introduction of a double strand break (DSB) at the mutational site by highly specific endonucleases, such as TALENs (transcription activator like effector nucleases) or RNA based nucleases (clustered regulatory interspaced short palindromic repeats - CRISPR-Cas), the cell's own DNA repair machinery restores integrity to the DNA strand and corrects the mutant sequence, thus allowing the cell to produce protein levels as needed...
September 10, 2016: Progress in Retinal and Eye Research
https://www.readbyqxmd.com/read/27575535/mar-mediated-transgene-integration-into-permissive-chromatin-and-increased-expression-by-recombination-pathway-engineering
#7
Kaja Kostyrko, Samuel Neuenschwander, Thomas Junier, Alexandre Regamey, Christian Iseli, Emanuel Schmid-Siegert, Sandra Bosshard, Stefano Majocchi, Valérie Le Fourn, Pierre-Alain Girod, Ioannis Xenarios, Nicolas Mermod
Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect...
August 29, 2016: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/27543296/rapid-screening-for-crispr-directed-editing-of-the-drosophila-genome-using-white-coconversion
#8
Daniel Tianfang Ge, Cindy Tipping, Michael H Brodsky, Phillip D Zamore
Adoption of a streamlined version of the bacterial clustered regular interspersed short palindromic repeat (CRISPR)/Cas9 defense system has accelerated targeted genome engineering. The Streptococcus pyogenes Cas9 protein, directed by a simplified, CRISPR-like single-guide RNA, catalyzes a double-stranded DNA break at a specific genomic site; subsequent repair by end joining can introduce mutagenic insertions or deletions, while repair by homologous recombination using an exogenous DNA template can incorporate new sequences at the target locus...
October 13, 2016: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/27537368/the-transcriptional-response-to-dna-double-strand-breaks-in-physcomitrella-patens
#9
Yasuko Kamisugi, John W Whitaker, Andrew C Cuming
The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting...
2016: PloS One
https://www.readbyqxmd.com/read/27494941/in-vitro-model-for-dna-double-strand-break-repair-analysis-in-breast-cancer-reveals-cell-type-specific-associations-with-age-and-prognosis
#10
Miriam Deniz, Julia Kaufmann, Andreea Stahl, Theresa Gundelach, Wolfgang Janni, Isabell Hoffmann, Marlen Keimling, Stephanie Hampp, Michaela Ihle, Lisa Wiesmüller
Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients)...
August 5, 2016: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/27492056/induction-of-dna-damage-and-erroneous-repair-can-explain-genomic-instability-caused-by-endosulfan
#11
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
https://www.readbyqxmd.com/read/27453047/essential-roles-for-polymerase-%C3%AE-mediated-end-joining-in-the-repair-of-chromosome-breaks
#12
David W Wyatt, Wanjuan Feng, Michael P Conlin, Matthew J Yousefzadeh, Steven A Roberts, Piotr Mieczkowski, Richard D Wood, Gaorav P Gupta, Dale A Ramsden
DNA polymerase theta (Pol θ)-mediated end joining (TMEJ) has been implicated in the repair of chromosome breaks, but its cellular mechanism and role relative to canonical repair pathways are poorly understood. We show that it accounts for most repairs associated with microhomologies and is made efficient by coupling a microhomology search to removal of non-homologous tails and microhomology-primed synthesis across broken ends. In contrast to non-homologous end joining (NHEJ), TMEJ efficiently repairs end structures expected after aborted homology-directed repair (5' to 3' resected ends) or replication fork collapse...
August 18, 2016: Molecular Cell
https://www.readbyqxmd.com/read/27314941/chromosomal-translocations-in-the-parasite-leishmania-by-a-mre11-rad50-independent-microhomology-mediated-end-joining-mechanism
#13
Marie-Claude N Laffitte, Philippe Leprohon, Maripier Hainse, Danielle Légaré, Jean-Yves Masson, Marc Ouellette
The parasite Leishmania often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in Leishmania would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate RAD50 null mutants in a wild-type background failed and we provide evidence that this gene is essential...
June 2016: PLoS Genetics
https://www.readbyqxmd.com/read/27264557/dna-polymerase-%C3%AE-polq-double-strand-break-repair-and-cancer
#14
REVIEW
Richard D Wood, Sylvie Doublié
DNA polymerase theta (pol θ) is encoded in the genomes of many eukaryotes, though not in fungi. Pol θ is encoded by the POLQ gene in mammalian cells. The C-terminal third of the protein is a family A DNA polymerase with additional insertion elements relative to prokaryotic homologs. The N-terminal third is a helicase-like domain with DNA-dependent ATPase activity. Pol θ is important in the repair of genomic double-strand breaks (DSBs) from many sources. These include breaks formed by ionizing radiation and topoisomerase inhibitors, breaks arising at stalled DNA replication forks, breaks introduced during diversification steps of the mammalian immune system, and DSB induced by CRISPR-Cas9...
August 2016: DNA Repair
https://www.readbyqxmd.com/read/27131361/a-role-for-human-homologous-recombination-factors-in-suppressing-microhomology-mediated-end-joining
#15
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
https://www.readbyqxmd.com/read/27113385/identification-of-dna-double-strand-breaks-at-chromosome-boundaries-along-the-track-of-particle-irradiation
#16
Atsuko Niimi, Motohiro Yamauchi, Siripan Limsirichaikul, Ryota Sekine, Takahiro Oike, Hiro Sato, Keiji Suzuki, Kathryn D Held, Takashi Nakano, Atsushi Shibata
Chromosomal translocations arise from misrejoining of DNA double strand breaks (DSBs) between loci located on two chromosomes. One current model suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. Ionizing radiation (IR) is a potent inducer of translocations. Accumulating evidence demonstrates that particle irradiation more frequently causes translocations compared with X-ray irradiation. This observation has led to the hypothesis that the high frequency of translocations after particle irradiation may be due to the formation of DSBs at chromosome boundaries along the particle track, because such DSBs can be misrejoined between distinct chromosomes...
August 2016: Genes, Chromosomes & Cancer
https://www.readbyqxmd.com/read/27109384/a-genetic-analysis-of-23-chinese-patients-with-hemophilia-b
#17
Qing-Yun Wang, Bei Hu, Hui Liu, Liang Tang, Wei Zeng, Ying-Ying Wu, Zhi-Peng Cheng, Yu Hu
Hemophilia B (HB) is an X-linked recessive bleeding disorder caused by mutations in the coagulation factor IX (FIX) gene. Genotyping patients with HB is essential for genetic counseling and provides useful information for patient management. In this study, the F9 gene from 23 patients with HB was analyzed by direct sequencing. Nineteen point mutations were identified, including a novel missense variant (c.520G > C, p.Val174Leu) in a patient with severe HB and a previously unreported homozygous missense mutation (c...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27063650/ligase-4-deficiency-causes-distinctive-immune-abnormalities-in-asymptomatic-individuals
#18
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
https://www.readbyqxmd.com/read/27058754/the-dna-resection-protein-ctip-promotes-mammary-tumorigenesis
#19
Colleen R Reczek, Reena Shakya, Yana Miteva, Matthias Szabolcs, Thomas Ludwig, Richard Baer
Many DNA repair factors act to suppress tumor formation by preserving genomic stability. Similarly, the CtIP protein, which interacts with the BRCA1 tumor suppressor, is also thought to have tumor suppression activity. Through its role in DNA end resection, CtIP facilitates DNA double-strand break (DSB) repair by homologous recombination (DSBR-HR) and microhomology-mediated end joining (MMEJ). In addition, however, CtIP has also been implicated in the formation of aberrant chromosomal rearrangements in an MMEJ-dependent manner, an activity that could potentially promote tumor development by increasing genome instability...
May 31, 2016: Oncotarget
https://www.readbyqxmd.com/read/26790771/risky-business-microhomology-mediated-end-joining
#20
Supriya Sinha, Diana Villarreal, Eun Yong Shim, Sang Eun Lee
Prevalence of microhomology (MH) at the breakpoint junctions in somatic and germ-line chromosomal rearrangements and in the programmed immune receptor rearrangements from cells deficient in classical end joining reveals an enigmatic process called MH-mediated end joining (MMEJ). MMEJ repairs DNA double strand breaks (DSBs) by annealing flanking MH and deleting genetic information at the repair junctions from yeast to humans. Being genetically distinct from canonical DNA DSB pathways, MMEJ is involved with the fusions of eroded/uncapped telomeres as well as with the assembly of chromosome fragments in chromothripsis...
June 2016: Mutation Research
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