scholarly journals Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation

Blood ◽  
2009 ◽  
Vol 113 (13) ◽  
pp. 2965-2975 ◽  
Author(s):  
William Giblin ◽  
Monalisa Chatterji ◽  
Gerwin Westfield ◽  
Tehmina Masud ◽  
Brian Theisen ◽  
...  
Keyword(s):  
Immune System ◽  
Dna Cleavage ◽  
Severe Combined Immunodeficiency ◽  
Double Strand Breaks ◽  
Mutant Form ◽  
Dna Rearrangements ◽  
Strand Breaks ◽  
Immune System Dysfunction

Abstract The RAG1/2 endonuclease initiates programmed DNA rearrangements in progenitor lymphocytes by generating double-strand breaks at specific recombination signal sequences. This process, known as V(D)J recombination, assembles the vastly diverse antigen receptor genes from numerous V, D, and J coding segments. In vitro biochemical and cellular transfection studies suggest that RAG1/2 may also play postcleavage roles by forming complexes with the recombining ends to facilitate DNA end processing and ligation. In the current study, we examine the in vivo consequences of a mutant form of RAG1, RAG1-S723C, that is proficient for DNA cleavage, yet exhibits defects in postcleavage complex formation and end joining in vitro. We generated a knockin mouse model harboring the RAG1-S723C hypomorphic mutation and examined the immune system in this fully in vivo setting. RAG1-S723C homozygous mice exhibit impaired lymphocyte development and decreased V(D)J rearrangements. Distinct from RAG nullizygosity, the RAG1-S723C hypomorph results in aberrant DNA double-strand breaks within rearranging loci. RAG1-S723C also predisposes to thymic lymphomas associated with chromosomal translocations in a p53 mutant background, and heterozygosity for the mutant allele accelerates age-associated immune system dysfunction. Thus, our study provides in vivo evidence that implicates aberrant RAG1/2 activity in lymphoid tumor development and premature immunosenescence.

scholarly journals A Role for DEAD Box 1 at DNA Double-Strand Breaks

2008 ◽  
Vol 28 (20) ◽  
pp. 6413-6425 ◽  
Author(s):  
Lei Li ◽  
Elizabeth A. Monckton ◽  
Roseline Godbout
Keyword(s):  
Ionizing Radiation ◽  
Neuroblastoma Cell ◽  
Rnase H ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Rna Helicases ◽  
Strand Breaks ◽  
Dead Box

ABSTRACT DEAD box proteins are a family of putative RNA helicases associated with all aspects of cellular metabolism involving the modification of RNA secondary structure. DDX1 is a member of the DEAD box protein family that is overexpressed in a subset of retinoblastoma and neuroblastoma cell lines and tumors. DDX1 is found primarily in the nucleus, where it forms two to four large aggregates called DDX1 bodies. Here, we report a rapid redistribution of DDX1 in cells exposed to ionizing radiation, resulting in the formation of numerous foci that colocalize with γ-H2AX and phosphorylated ATM foci at sites of DNA double-strand breaks (DSBs). The formation of DDX1 ionizing-radiation-induced foci (IRIF) is dependent on ATM, which was shown to phosphorylate DDX1 both in vitro and in vivo. The treatment of cells with RNase H prevented the formation of DDX1 IRIF, suggesting that DDX1 is recruited to sites of DNA damage containing RNA-DNA structures. We have shown that DDX1 has RNase activity toward single-stranded RNA, as well as ADP-dependent RNA-DNA- and RNA-RNA-unwinding activities. We propose that DDX1 plays an RNA clearance role at DSB sites, thereby facilitating the template-guided repair of transcriptionally active regions of the genome.

scholarly journals A Domesticated piggyBac Transposase Plays Key Roles in Heterochromatin Dynamics and DNA Cleavage during Programmed DNA Deletion in Tetrahymena thermophila

2010 ◽  
Vol 21 (10) ◽  
pp. 1753-1762 ◽  
Author(s):  
Chao-Yin Cheng ◽  
Alexander Vogt ◽  
Kazufumi Mochizuki ◽  
Meng-Chao Yao
Keyword(s):  
Dna Cleavage ◽  
Double Strand Breaks ◽  
Cellular Functions ◽  
Transposase Gene ◽  
Strand Breaks ◽  
Dna Deletion ◽  
Final Assembly ◽  
Dna Elimination ◽  
Cleavage Step

Transposons comprise large fractions of eukaryotic genomes and provide genetic reservoirs for the evolution of new cellular functions. We identified TPB2, a homolog of the piggyBac transposase gene that is required for programmed DNA deletion in Tetrahymena. TPB2 was expressed exclusively during the time of DNA excision, and its encoded protein Tpb2p was localized in DNA elimination heterochromatin structures. Notably, silencing of TPB2 by RNAi disrupts the final assembly of these heterochromatin structures and prevents DNA deletion to occur. In vitro studies revealed that Tpb2p is an endonuclease that produces double-strand breaks with four-base 5′ protruding ends, similar to the ends generated during DNA deletion. These findings suggest that Tpb2p plays a key role in the assembly of specialized DNA elimination chromatin architectures and is likely responsible for the DNA cleavage step of programmed DNA deletion.

scholarly journals Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration

2019 ◽  
Vol 1 (1) ◽  
Author(s):  
Richard I Tuxworth ◽  
Matthew J Taylor ◽  
Ane Martin Anduaga ◽  
Alaa Hussien-Ali ◽  
Sotiroula Chatzimatthaiou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nervous System ◽  
Dna Damage Response ◽  
Neurological Disorders ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Ganglion Neurons

Abstract DNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double-strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective. Here, we show that attenuating the DNA damage response by targeting the meiotic recombination 11, Rad50, Nijmegen breakage syndrome 1 complex, which is involved in double-strand break recognition, is neuroprotective in three neurodegeneration models in Drosophila and prevents Aβ1-42-induced loss of synapses in embryonic hippocampal neurons. Attenuating the DNA damage response after optic nerve injury is also neuroprotective to retinal ganglion cells and promotes dramatic regeneration of their neurites both in vitro and in vivo. Dorsal root ganglion neurons similarly regenerate when the DNA damage response is targeted in vitro and in vivo and this strategy also induces significant restoration of lost function after spinal cord injury. We conclude that muting the DNA damage response in the nervous system is neuroprotective in multiple neurological disorders. Our results point to new therapies to maintain or repair the nervous system.

scholarly journals The rad51-K191R ATPase-Defective Mutant Is Impaired forPresynaptic Filament Formation

2006 ◽  
Vol 26 (24) ◽  
pp. 9544-9554 ◽  
Author(s):  
Cindy W. Fung ◽  
Gary S. Fortin ◽  
Shaun E. Peterson ◽  
Lorraine S. Symington
Keyword(s):  
Atp Hydrolysis ◽  
Double Strand Breaks ◽  
Strand Exchange ◽  
Filament Formation ◽  
Homologous Pairing ◽  
Strand Breaks ◽  
Defective Mutant ◽  
Srs2 Helicase

ABSTRACT The nucleoprotein filament formed by Rad51 polymerization on single-stranded DNA is essential for homologous pairing and strand exchange. ATP binding is required for Rad51 nucleoprotein filament formation and strand exchange, but ATP hydrolysis is not required for these functions in vitro. Previous studies have shown that a yeast strain expressing the rad51-K191R allele is sensitive to ionizing radiation, suggesting an important role for ATP hydrolysis in vivo. The recruitment of Rad51-K191R to double-strand breaks is defective in vivo, and this phenotype can be suppressed by elimination of the Srs2 helicase, an antagonist of Rad51 filament formation. The phenotype of the rad51-K191R strain is also suppressed by overexpression of Rad54. In vitro, the Rad51-K191R protein exhibits a slight decrease in binding to DNA, consistent with the defect in presynaptic filament formation. However, the rad51-K191R mutation is dominant in heterozygous diploids, indicating that the defect is not due simply to reduced affinity for DNA. We suggest the Rad51-K191R protein either forms an altered filament or is defective in turnover, resulting in a reduced pool of free protein available for DNA binding.

scholarly journals Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model.

1986 ◽  
Vol 6 (11) ◽  
pp. 3831-3837 ◽  
Author(s):  
M Jayaram
Keyword(s):  
Mating Type ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Site Specific ◽  
Strand Breaks ◽  
Conversion Event ◽  
Yeast Mating ◽  
Mating Type Switching

Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.

scholarly journals Attenuating the DNA damage response to double strand breaks restores function in models of CNS neurodegeneration

2018 ◽  
Author(s):  
Richard I. Tuxworth ◽  
Matthew J. Taylor ◽  
Ane Martin Anduaga ◽  
Alaa Hussien-Ali ◽  
Sotiroula Chatzimatthaiou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nervous System ◽  
Dna Damage Response ◽  
Neurological Disorders ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Ganglion Neurons

AbstractDNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective. Here, we show that attenuating the DNA damage response by targeting the meiotic recombination 11, Rad50, Nijmegen breakage syndrome 1 complex, which is involved in double strand break recognition, is neuroprotective in three neurodegeneration models in Drosophila and prevents Aβ1-42-induced loss of synapses in embryonic hippocampal neurons. Attenuating the DNA damage response after optic nerve injury is also neuroprotective to retinal ganglion cells and promotes dramatic regeneration of their neurites both in vitro and in vivo. Dorsal root ganglion neurons similarly regenerate when the DNA damage response is targeted in vitro and in vivo and this strategy also induces significant restoration of lost function after spinal cord injury. We conclude that muting the DNA damage response in the nervous system is neuroprotective in multiple neurological disorders. Our results point to new therapies to maintain or repair the nervous system.

scholarly journals High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

2020 ◽  
Author(s):  
Karthik Murugan ◽  
Arun S. Seetharam ◽  
Andrew J. Severin ◽  
Dipali G. Sashital
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Double Strand Breaks ◽  
Target Sequence ◽  
Wild Type ◽  
Strand Breaks ◽  
Cleavage Assay ◽  
Target Sequences

AbstractCas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wild-type (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high-throughput in vitro cleavage assay to compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9, and HiFi Cas9). We observed that all Cas9s tested were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.

scholarly journals The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions

2018 ◽  
Vol 12 (11) ◽  
pp. e0006875 ◽  
Author(s):  
Danielle Gomes Passos Silva ◽  
Selma da Silva Santos ◽  
Sheila C. Nardelli ◽  
Isabela Cecília Mendes ◽  
Anna Cláudia Guimarães Freire ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

scholarly journals Visualization of Repair of Double-Strand Breaks in the Bacteriophage T7 Genome without Normal DNA Replication

2000 ◽  
Vol 182 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Ying-Ta Lai ◽  
Warren Masker
Keyword(s):  
Dna Synthesis ◽  
High Efficiency ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Bacteriophage T7 ◽  
Growth Step ◽  
Strand Breaks

ABSTRACT An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 is able to repair double-strand breaks in a T7 genome with efficiencies of 20% or more. To achieve this high repair efficiency it is necessary that the reaction mixtures contain molecules of donor DNA that bracket the double-strand break. Gaps as long as 1,600 nucleotides are repaired almost as efficiently as simple double-strand breaks. DNA synthesis was measured while repair was taking place. It was found that the amount of DNA synthesis associated with repair of a double-strand break was below the level of detection possible with this system. Furthermore, repair efficiencies were the same with or without normal levels of T7 DNA polymerase. However, the repair required the 5′→3′ exonuclease encoded by T7 gene 6. The high efficiency of DNA repair allowed visualization of the repaired product after in vitro repair, thereby assuring that the repair took place in vitro rather than during an in vivo growth step after packaging.

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