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microhomology mediated end join

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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Chi Zhang, Xiuhua Meng, Xiaolei Wei, Ling Lu
Filamentous fungi have a dominant nonhomologous-end joining (NHEJ) DNA repair pathway, which results in the majority of transformed progenies having random heterologous insertion mutagenesis. Thus, lack of a versatile genome-editing tool prevents us from carrying out precise genome editing to explore the mechanism of pathogenesis. Moreover, clinical isolates that have a wild-type ku80 background without any selection nutrition marker especially suffer from low homologous integration efficiency. In this study, we have established a highly efficient CRISPR mutagenesis system to carry out precise and efficient in-frame integration with or without marker insertion with approximately 95-100% accuracy via very short (approximately 35-bp) homology arms in a process referred to as microhomology-mediated end joining (MMEJ)...
January 2016: Fungal Genetics and Biology: FG & B
Tetsushi Sakuma, Shota Nakade, Yuto Sakane, Ken-Ichi T Suzuki, Takashi Yamamoto
Programmable nucleases enable engineering of the genome by utilizing endogenous DNA double-strand break (DSB) repair pathways. Although homologous recombination (HR)-mediated gene knock-in is well established, it cannot necessarily be applied in every cell type and organism because of variable HR frequencies. We recently reported an alternative method of gene knock-in, named the PITCh (Precise Integration into Target Chromosome) system, assisted by microhomology-mediated end-joining (MMEJ). MMEJ harnesses independent machinery from HR, and it requires an extremely short homologous sequence (5-25 bp) for DSB repair, resulting in precise gene knock-in with a more easily constructed donor vector...
January 2016: Nature Protocols
Susanne Lorenz, Tale Barøy, Jinchang Sun, Torfinn Nome, Daniel Vodák, Jan-Christian Bryne, Anne-Mari Håkelien, Lynnette Fernandez-Cuesta, Birte Möhlendick, Harald Rieder, Karoly Szuhai, Olga Zaikova, Terje C Ahlquist, Gard O S Thomassen, Rolf I Skotheim, Ragnhild A Lothe, Patrick S Tarpey, Peter Campbell, Adrienne Flanagan, Ola Myklebost, Leonardo A Meza-Zepeda
In contrast to many other sarcoma subtypes, the chaotic karyotypes of osteosarcoma have precluded the identification of pathognomonic translocations. We here report hundreds of genomic rearrangements in osteosarcoma cell lines, showing clear characteristics of microhomology-mediated break-induced replication (MMBIR) and end-joining repair (MMEJ) mechanisms. However, at RNA level, the majority of the fused transcripts did not correspond to genomic rearrangements, suggesting the involvement of trans-splicing, which was further supported by typical trans-splicing characteristics...
February 2, 2016: Oncotarget
Kaja Kostyrko, Nicolas Mermod
DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms...
April 7, 2016: Nucleic Acids Research
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