CRISPR-Cas targeted plasmid integration into mammalian cells via non-homologous end joining

2015 ◽  
Vol 112 (10) ◽  
pp. 2154-2162 ◽  
Author(s):  
Ravichandra Bachu ◽  
Iñigo Bergareche ◽  
Lawrence A. Chasin
Keyword(s):  
Mammalian Cells ◽  
End Joining ◽  
Plasmid Integration ◽  
Non Homologous End Joining

scholarly journals Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
Lalith Perera ◽  
David D. Shock ◽  
William A. Beard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Strand Break ◽  
Structural Basis ◽  
End Joining ◽  
Strand Breaks ◽  
Nucleotide Pools ◽  
Nucleotide Insertion ◽  
Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

scholarly journals DNA repair of clustered lesions in mammalian cells: involvement of non-homologous end-joining

2008 ◽  
Vol 36 (15) ◽  
pp. 4872-4882 ◽  
Author(s):  
S. Malyarchuk ◽  
R. Castore ◽  
L. Harrison
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
End Joining ◽  
Non Homologous End Joining

scholarly journals Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells

2010 ◽  
Vol 285 (30) ◽  
pp. 23198-23207 ◽  
Author(s):  
Jia Liu ◽  
Alokes Majumdar ◽  
Jilan Liu ◽  
Lawrence H. Thompson ◽  
Michael M. Seidman
Keyword(s):  
Mammalian Cells ◽  
Single Strand ◽  
End Joining ◽  
Non Homologous End Joining

DNA double-strand break repair within heterochromatic regions

2012 ◽  
Vol 40 (1) ◽  
pp. 173-178 ◽  
Author(s):  
Johanne M. Murray ◽  
Tom Stiff ◽  
Penny A. Jeggo
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
Strand Break ◽  
Higher Order ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Dna Dsbs ◽  
A Cell ◽  
Non Homologous End Joining

DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.

scholarly journals Ku-dependent non-homologous end-joining as the major pathway contributes to sublethal damage repair in mammalian cells

2015 ◽  
Vol 91 (11) ◽  
pp. 867-871 ◽  
Author(s):  
Min Liu ◽  
Solah Lee ◽  
Bailong Liu ◽  
Hongyan Wang ◽  
Lihua Dong ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
End Joining ◽  
Major Pathway ◽  
Damage Repair ◽  
Non Homologous End Joining

scholarly journals DNA Substrate Dependence of p53-Mediated Regulation of Double-Strand Break Repair

2002 ◽  
Vol 22 (17) ◽  
pp. 6306-6317 ◽  
Author(s):  
Nuray Akyüz ◽  
Gisa S. Boehden ◽  
Silke Süsse ◽  
Andreas Rimek ◽  
Ute Preuss ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
P53 Expression ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Multistep Process ◽  
Non Homologous End Joining

ABSTRACT DNA double-strand breaks (DSBs) arise spontaneously after the conversion of DNA adducts or single-strand breaks by DNA repair or replication and can be introduced experimentally by expression of specific endonucleases. Correct repair of DSBs is central to the maintenance of genomic integrity in mammalian cells, since errors give rise to translocations, deletions, duplications, and expansions, which accelerate the multistep process of tumor progression. For p53 direct regulatory roles in homologous recombination (HR) and in non-homologous end joining (NHEJ) were postulated. To systematically analyze the involvement of p53 in DSB repair, we generated a fluorescence-based assay system with a series of episomal and chromosomally integrated substrates for I-SceI meganuclease-triggered repair. Our data indicate that human wild-type p53, produced either stably or transiently in a p53-negative background, inhibits HR between substrates for conservative HR (cHR) and for gene deletions. NHEJ via microhomologies flanking the I-SceI cleavage site was also downregulated after p53 expression. Interestingly, the p53-dependent downregulation of homology-directed repair was maximal during cHR between sequences with short homologies. Inhibition was minimal during recombination between substrates that support reporter gene reconstitution by HR and NHEJ. p53 with a hotspot mutation at codon 281, 273, 248, 175, or 143 was severely defective in regulating DSB repair (frequencies elevated up to 26-fold). For the transcriptional transactivation-inactive variant p53(138V) a defect became apparent with short homologies only. These results suggest that p53 plays a role in restraining DNA exchange between imperfectly homologous sequences and thereby in suppressing tumorigenic genome rearrangements.

scholarly journals Non-Homologous end Joining Factors XLF, PAXX and DNA-PKcs Maintain the Neural Stem and Progenitor Cell Population

2020 ◽  
Author(s):  
Raquel Gago-Fuentes ◽  
Valentyn Oksenych
Keyword(s):  
Mammalian Cells ◽  
Progenitor Cell ◽  
Neuronal Apoptosis ◽  
Embryonic Lethality ◽  
End Joining ◽  
Double Knockout ◽  
Dna Damages ◽  
Stem And Progenitor Cells ◽  
Non Homologous End Joining ◽  
The Impact

Non-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damages throughout the cell cycle, and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking NHEJ factors Ku70 and Ku80. Inactivation of other NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of either Paxx, Mri or Dna-pkcs NHEJ gene results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of NHEJ factors on early stages of neurodevelopment, we isolated neural stem and progenitor cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf, Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that XLF, DNA-PKcs and PAXX maintain the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models.

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