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 Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

2002 ◽  
Vol 195 (3) ◽  
pp. 309-316 ◽  
Author(s):  
Robert E. Tillman ◽  
Andrea L. Wooley ◽  
Maureen M. Hughes ◽  
Tara D. Wehrly ◽  
Wojciech Swat ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Receptor Gene ◽  
Antigen Receptor ◽  
Double Strand Breaks ◽  
Recombination Reaction ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Signal Sequences ◽  
Dna Dsbs ◽  
Cleavage Step

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.

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 Functions of the CSB Protein at Topoisomerase 2 Inhibitors-Induced DNA Lesions

2021 ◽  
Vol 9 ◽  
Author(s):  
Franciele Faccio Busatto ◽  
Sofiane Y. Mersaoui ◽  
Yilun Sun ◽  
Yves Pommier ◽  
Jean-Yves Masson ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Excision Repair ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Nucleotide Excision ◽  
Different Types ◽  
Topoisomerase 2 ◽  
Nucleotide Excision Repair Pathway

Topoisomerase 2 (TOP2) inhibitors are drugs widely used in the treatment of different types of cancer. Processing of their induced-lesions create double-strand breaks (DSBs) in the DNA, which is the main toxic mechanism of topoisomerase inhibitors to kill cancer cells. It was established that the Nucleotide Excision Repair pathway respond to TOP2-induced lesions, mainly through the Cockayne Syndrome B (CSB) protein. In this paper, we further define the mechanism and type of lesions induced by TOP2 inhibitors when CSB is abrogated. In the absence of TOP2, but not during pharmacological inhibition, an increase in R-Loops was detected. We also observed that CSB knockdown provokes the accumulation of DSBs induced by TOP2 inhibitors. Consistent with a functional interplay, interaction between CSB and TOP2 occurred after TOP2 inhibition. This was corroborated with in vitro DNA cleavage assays where CSB stimulated the activity of TOP2. Altogether, our results show that TOP2 is stimulated by the CSB protein and prevents the accumulation of R-loops/DSBs linked to genomic instability.

Bestimmung der DNA-Schädigung in vitro

2010 ◽  
Vol 49 (S 01) ◽  
pp. S64-S68
Author(s):  
E. Dikomey
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Chromosome Aberrations ◽  
Genetic Factors ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Cell Inactivation ◽  
Repair Mechanisms ◽  
Break Repair

SummaryIonising irradiation acts primarily via induction of DNA damage, among which doublestrand breaks are the most important lesions. These lesions may lead to lethal chromosome aberrations, which are the main reason for cell inactivation. Double-strand breaks can be repaired by several different mechanisms. The regulation of these mechanisms appears be fairly different for normal and tumour cells. Among different cell lines capacity of doublestrand break repair varies by only few percents and is known to be determined mostly by genetic factors. Knowledge about doublestrand break repair mechanisms and their regulation is important for the optimal application of ionising irradiation in medicine.

scholarly journals Focused ultrasound radiosensitizes human cancer cells by enhancement of DNA damage

2021 ◽  
Author(s):  
Xinrui Zhang ◽  
Mariana Bobeica ◽  
Michael Unger ◽  
Anastasia Bednarz ◽  
Bjoern Gerold ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Metabolic Activity ◽  
Focused Ultrasound ◽  
Double Strand Breaks ◽  
High Intensity Focused Ultrasound ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Abstract Purpose High-intensity focused ultrasound (HIFU/FUS) has expanded as a noninvasive quantifiable option for hyperthermia (HT). HT in a temperature range of 40–47 °C (thermal dose CEM43 ≥ 25) could work as a sensitizer to radiation therapy (RT). Here, we attempted to understand the tumor radiosensitization effect at the cellular level after a combination treatment of FUS+RT. Methods An in vitro FUS system was developed to induce HT at frequencies of 1.147 and 1.467 MHz. Human head and neck cancer (FaDU), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS in ultrasound-penetrable 96-well plates followed by single-dose X‑ray irradiation (10 Gy). Radiosensitizing effects of FUS were investigated by cell metabolic activity (WST‑1 assay), apoptosis (annexin V assay, sub-G1 assay), cell cycle phases (propidium iodide staining), and DNA double-strand breaks (γH2A.X assay). Results The FUS intensities of 213 (1.147 MHz) and 225 W/cm2 (1.467 MHz) induced HT for 30 min at mean temperatures of 45.20 ± 2.29 °C (CEM43 = 436 ± 88) and 45.59 ± 1.65 °C (CEM43 = 447 ± 79), respectively. FUS improves the effect of RT significantly by reducing metabolic activity in T98G cells 48 h (RT: 96.47 ± 8.29%; FUS+RT: 79.38 ± 14.93%; p = 0.012) and in PC-3 cells 72 h (54.20 ± 10.85%; 41.01 ± 11.17%; p = 0.016) after therapy, but not in FaDu cells. Mechanistically, FUS+RT leads to increased apoptosis and enhancement of DNA double-strand breaks compared to RT alone in T98G and PC-3 cells. Conclusion Our in vitro findings demonstrate that FUS has good potential to sensitize glioblastoma and prostate cancer cells to RT by mainly enhancing DNA damage.

scholarly journals Double-strand DNA breaks recruit the centromeric histone CENP-A

2009 ◽  
Vol 106 (37) ◽  
pp. 15762-15767 ◽  
Author(s):  
Samantha G. Zeitlin ◽  
Norman M. Baker ◽  
Brian R. Chapados ◽  
Evi Soutoglou ◽  
Jean Y. J. Wang ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
Histone H3 Variant ◽  
Radiation Induced ◽  
Non Homologous End Joining

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

scholarly journals In Vitro Evaluation of No-carrier-added Radiolabeled Cisplatin ([189, 191Pt]cisplatin) Emitting Auger Electrons

2021 ◽  
Author(s):  
Honoka Obata ◽  
Atsushi B. Tsuji ◽  
Hitomi Sudo ◽  
Aya Sugyo ◽  
Katsuyuki Minegishi ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Short Range ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Primary Target ◽  
Auger Electrons ◽  
Strand Breaks ◽  
Chemical Damage ◽  
No Carrier Added

Due to their short range (2–500 nm), Auger electrons (Auger e-) have the potential to induce nano-scale physiochemical damage to biomolecules. Although DNA is the primary target of Au-ger e-, it remains challenging to maximize the interaction between Auger e- and DNA. To assess the DNA-damaging effect of Auger e- released as close as possible to DNA without chemical damage, we radio-synthesized no-carrier-added (n.c.a.) [189, 191Pt]cisplatin and evaluated both its in vitro properties and DNA-damaging effect. Cellular uptake, intracellular distribution, and DNA binding were investigated, and DNA double-strand breaks (DSBs) were evaluated by im-munofluorescence staining of γH2AX and gel electrophoresis of plasmid DNA. Approximately 20% of intracellular radio-Pt was in a nucleus, and about 2% of intra-nucleus radio-Pt bound to DNA, although uptake of n.c.a. radio-cisplatin was low (0.6% incubated dose after 25-h incuba-tion), resulting in the frequency of cells with γH2AX foci was low (1%). Nevertheless, some cells treated with radio-cisplatin had γH2AX aggregates unlike non-radioactive cisplatin. These findings suggest n.c.a. radio-cisplatin binding to DNA causes severe DSBs by release of Auger e- very close to DNA without chemical damage by carriers. Efficient radio-drug delivery to DNA is necessary for successful clinical application of Auger e-.

scholarly journals TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

2021 ◽  
Author(s):  
Alexandre Nore ◽  
Ariadna B Juarez-Martinez ◽  
Julie AJ Clement ◽  
Christine Brun ◽  
Bouboub Diagouraga ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Point Mutations ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Catalytic Complex ◽  
Strand Breaks ◽  
Genome Wide ◽  
Dna Cleavage Activity ◽  
Meiotic Dsbs

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. In male and female meiotic cells, DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identified their conserved interacting domains by structural analysis. We then analysed the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity was strongly reduced genome-wide in oocytes, but only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity was delayed in autosomes. These results provide evidence that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity by integrating specific chromosomal features.

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