scholarly journals Visualization of complex DNA damage along accelerated ions tracks

2018 ◽  
Vol 177 ◽  
pp. 06002
Author(s):  
Elena Kulikova ◽  
Alla Boreyko ◽  
Tatiana Bulanova ◽  
Lucie Ježková ◽  
Mariia Zadneprianetc ◽  
...  
Keyword(s):  
Dna Damage ◽  
Human Fibroblasts ◽  
Dna Lesions ◽  
3D Analysis ◽  
Dna Double Strand Breaks ◽  
Base Damage ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Dna Dsbs ◽  
Γ Rays

The most deleterious DNA lesions induced by ionizing radiation are clustered DNA double-strand breaks (DSB). Clustered or complex DNA damage is a combination of a few simple lesions (single-strand breaks, base damage etc.) within one or two DNA helix turns. It is known that yield of complex DNA lesions increases with increasing linear energy transfer (LET) of radiation. For investigation of the induction and repair of complex DNA lesions, human fibroblasts were irradiated with high-LET 15N ions (LET = 183.3 keV/μm, E = 13MeV/n) and low-LET 60Co γ-rays (LET ≈ 0.3 keV/μm) radiation. DNA DSBs (γH2AX and 53BP1) and base damage (OGG1) markers were visualized by immunofluorecence staining and high-resolution microscopy. The obtained results showed slower repair kinetics of induced DSBs in cells irradiated with accelerated ions compared to 60Co γ-rays, indicating induction of more complex DNA damage. Confirming previous assumptions, detailed 3D analysis of γH2AX/53BP1 foci in 15N ions tracks revealed more complicated structure of the foci in contrast to γ-rays. It was shown that proteins 53BP1 and OGG1 involved in repair of DNA DSBs and modified bases, respectively, were colocalized in tracks of 15N ions and thus represented clustered DNA DSBs.

scholarly journals Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

2021 ◽  
Vol 22 (14) ◽  
pp. 7638
Author(s):  
Yvonne Lorat ◽  
Judith Reindl ◽  
Anna Isermann ◽  
Christian Rübe ◽  
Anna A. Friedl ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dna Damage ◽  
Spatial Clustering ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Stimulated Emission ◽  
Nuclear Chromatin ◽  
Carbon Ions ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

scholarly journals Recent advances in the nucleolar responses to DNA double-strand breaks

2020 ◽  
Vol 48 (17) ◽  
pp. 9449-9461
Author(s):  
Lea Milling Korsholm ◽  
Zita Gál ◽  
Blanca Nieto ◽  
Oliver Quevedo ◽  
Stavroula Boukoura ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Ribosomal Dna ◽  
Repetitive Sequences ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

scholarly journals Tetrahymena Meiotic Nuclear Reorganization Is Induced by a Checkpoint Kinase–dependent Response to DNA Damage

2009 ◽  
Vol 20 (9) ◽  
pp. 2428-2437 ◽  
Author(s):  
Josef Loidl ◽  
Kazufumi Mochizuki
Keyword(s):  
Dna Damage ◽  
Kinase Inhibitors ◽  
Signal Transduction Pathway ◽  
Dna Lesions ◽  
Meiotic Pairing ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Nuclear Reorganization ◽  
Bivalent Formation ◽  
Atr Kinase

In the ciliate Tetrahymena, meiotic micronuclei (MICs) undergo extreme elongation, and meiotic pairing and recombination take place within these elongated nuclei (the “crescents”). We have previously shown that elongation does not occur in the absence of Spo11p-induced DNA double-strand breaks (DSBs). Here we show that elongation is restored in spo11Δ mutants by various DNA-damaging agents including ones that may not cause DSBs to a notable extent. MIC elongation following Spo11p-induced DSBs or artificially induced DNA lesions is probably a DNA-damage response mediated by a phosphokinase signal transduction pathway, since it is suppressed by the ATM/ATR kinase inhibitors caffeine and wortmannin and by knocking out Tetrahymena's ATR orthologue. MIC elongation occurs concomitantly with the movement of centromeres away from the telomeric pole of the MIC. This DNA damage–dependent reorganization of the MIC helps to arrange homologous chromosomes alongside each other but is not sufficient for exact pairing. Thus, Spo11p contributes to bivalent formation in two ways: by creating a favorable spatial disposition of homologues and by stabilizing pairing by crossovers. The polarized chromosome orientation inside the crescent resembles the conserved meiotic bouquet, and crescent and bouquet also share the putative function of aiding meiotic pairing. However, they are regulated differently because in Tetrahymena, DSBs are required for entering rather than exiting this stage.

scholarly journals SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Sílvia Carvalho ◽  
Alexandra C Vítor ◽  
Sreerama C Sridhara ◽  
Filipa B Martins ◽  
Ana C Raposo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Strand Break ◽  
Dna Lesions ◽  
Alternative Mechanism ◽  
Dna Double Strand Breaks ◽  
Cell Renal Cell Carcinoma ◽  
Strand Breaks ◽  
Dna Damage Signaling ◽  
Dna Double Strand Break ◽  
Recombination Repair

Histone modifications establish the chromatin states that coordinate the DNA damage response. In this study, we show that SETD2, the enzyme that trimethylates histone H3 lysine 36 (H3K36me3), is required for ATM activation upon DNA double-strand breaks (DSBs). Moreover, we find that SETD2 is necessary for homologous recombination repair of DSBs by promoting the formation of RAD51 presynaptic filaments. In agreement, SETD2-mutant clear cell renal cell carcinoma (ccRCC) cells displayed impaired DNA damage signaling. However, despite the persistence of DNA lesions, SETD2-deficient cells failed to activate p53, a master guardian of the genome rarely mutated in ccRCC and showed decreased cell survival after DNA damage. We propose that this novel SETD2-dependent role provides a chromatin bookmarking instrument that facilitates signaling and repair of DSBs. In ccRCC, loss of SETD2 may afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for additional genetic mutations in TP53.

scholarly journals Poly(ADP-ribosyl)ation mediates early phase histone eviction at DNA lesions

2020 ◽  
Vol 48 (6) ◽  
pp. 3001-3013 ◽  
Author(s):  
Guang Yang ◽  
Yibin Chen ◽  
Jiaxue Wu ◽  
Shih-Hsun Chen ◽  
Xiuhua Liu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Molecular Mechanism ◽  
Parp Inhibitor ◽  
Repair Process ◽  
Dna Lesions ◽  
Histone Chaperones ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Lesion ◽  
Damage Repair

Abstract Nucleosomal histones are barriers to the DNA repair process particularly at DNA double-strand breaks (DSBs). However, the molecular mechanism by which these histone barriers are removed from the sites of DNA damage remains elusive. Here, we have generated a single specific inducible DSB in the cells and systematically examined the histone removal process at the DNA lesion. We found that histone removal occurred immediately following DNA damage and could extend up to a range of few kilobases from the lesion. To examine the molecular mechanism underlying DNA damage-induced histone removal, we screened histone modifications and found that histone ADP-ribosylation was associated with histone removal at DNA lesions. PARP inhibitor treatment suppressed the immediate histone eviction at DNA lesions. Moreover, we examined histone chaperones and found that the FACT complex recognized ADP-ribosylated histones and mediated the removal of histones in response to DNA damage. Taken together, our results reveal a pathway that regulates early histone barrier removal at DNA lesions. It may also explain the mechanism by which PARP inhibitor regulates early DNA damage repair.

Change in DNA after Exposure to Hydrogen Atoms.

1969 ◽  
Vol 24 (12) ◽  
pp. 1565-1573 ◽  
Author(s):  
H. Jung, ◽  
U. Hagen, ◽  
M. Ullrich, ◽  
E. E. Petersen
Keyword(s):  
Hydrogen Bonds ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Hydrogen Atoms ◽  
Γ Irradiation ◽  
Base Damage ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Single Breaks

The action of hydrogen atoms — generated in an electrodeless high frequency gas discharge — on calf thymus DNA in aqueous solution was investigated. The loss of priming activity was compared with the appearance of single strand breaks in native and denatured DNA, double strand breaks, denatured zones, base damage and rupture of hydrogen bonds. The primary lesions after exposure to H atoms and gamma radiation, respectively, are single strand breaks and base damage. Double strand breaks originating from accumulation of single breaks, and rupture of hydrogen bonds caused by single breaks and base damage, were identified as secondary lesions. In relation to strand breaks arising from radical attack on the sugar-phosphate backbone of the DNA molecule, base damage is about 12.5 times more frequent after Η-exposure than after γ-irradiation. It is concluded from this observation, that single strand breaks are the predominant critical lesions responsible for the loss of the functional activity of DNA.

scholarly journals DNA damage and its repair in lymphocytes and thyroid nodule cells during radioiodine therapy in patients with hyperthyroidism

2006 ◽  
Vol 37 (3) ◽  
pp. 527-532 ◽  
Author(s):  
W Grzesiuk ◽  
J Nieminuszczy ◽  
M Kruszewski ◽  
T Iwanienko ◽  
M Plazińska ◽  
...  
Keyword(s):  
Dna Damage ◽  
Thyroid Nodule ◽  
Peripheral Blood Lymphocytes ◽  
Radioiodine Treatment ◽  
Base Damage ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Autonomous Thyroid Nodule ◽  
The Individual ◽  
Cellular Dna

Radioiodine treatment of hyperthyroid patients with autonomous thyroid nodule leads to cellular DNA damage not only in thyrocytes but also in peripheral blood lymphocytes. The purpose of this study was to evaluate DNA breakage and base damage in thyrocytes and lymphocytes in patients treated with 131-I. In all the patients thyroid scintiscan was performed using 131-I. Damage to DNA was estimated by comet assay. Samples were taken before radioiodine treatment, and 12 and 54 days afterwards. Our results indicate high diversity in the level of DNA damage among the individual patients. However, in all cases, after 54 days the level of DNA damage in lymphocytes was similar or even lower than that in the controls. In contrast, in hot nodule the DNA damage persisted until the 54th day after 131-I application. Differences in the type of DNA damage between thyrocytes and lymphocytes were also observed. In lymphocytes there was more base damage, whereas in thyrocytes single strand breaks prevailed. This may indicate different mechanisms of DNA damage induction and/or DNA repair.

scholarly journals DDB1 Maintains Genome Integrity through Regulation of Cdt1

2006 ◽  
Vol 26 (21) ◽  
pp. 7977-7990 ◽  
Author(s):  
Courtney A. Lovejoy ◽  
Kimberli Lock ◽  
Ashwini Yenamandra ◽  
David Cortez
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Genome Instability ◽  
Excision Repair ◽  
Dna Lesions ◽  
Cell Cycle Checkpoints ◽  
Dna Double Strand Breaks ◽  
Genome Maintenance ◽  
Strand Breaks ◽  
Ubiquitin Ligase Complex

ABSTRACT DDB1, a component of a Cul4A ubiquitin ligase complex, promotes nucleotide excision repair (NER) and regulates DNA replication. We have investigated the role of human DDB1 in maintaining genome stability. DDB1-depleted cells accumulate DNA double-strand breaks in widely dispersed regions throughout the genome and have activated ATM and ATR cell cycle checkpoints. Depletion of Cul4A yields similar phenotypes, indicating that an E3 ligase function of DDB1 is important for genome maintenance. In contrast, depletion of DDB2, XPA, or XPC does not cause activation of DNA damage checkpoints, indicating that defects in NER are not involved. One substrate of DDB1-Cul4A that is crucial for preventing genome instability is Cdt1. DDB1-depleted cells exhibit increased levels of Cdt1 protein and rereplication, despite containing other Cdt1 regulatory mechanisms. The rereplication, accumulation of DNA damage, and activation of checkpoint responses in DDB1-depleted cells require entry into S phase and are partially, but not completely, suppressed by codepletion of Cdt1. Therefore, DDB1 prevents DNA lesions from accumulating in replicating human cells, in part by regulating Cdt1 degradation.

scholarly journals The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks

2012 ◽  
Vol 199 (7) ◽  
pp. 1067-1081 ◽  
Author(s):  
Céline Courilleau ◽  
Catherine Chailleux ◽  
Alain Jauneau ◽  
Fanny Grimal ◽  
Sébastien Briois ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Remodeling ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Dna Damage Signaling ◽  
Dna Dsbs ◽  
Dependent Processes

DNA damage signaling and repair take place in a chromatin context. Consequently, chromatin-modifying enzymes, including adenosine triphosphate–dependent chromatin remodeling enzymes, play an important role in the management of DNA double-strand breaks (DSBs). Here, we show that the p400 ATPase is required for DNA repair by homologous recombination (HR). Indeed, although p400 is not required for DNA damage signaling, DNA DSB repair is defective in the absence of p400. We demonstrate that p400 is important for HR-dependent processes, such as recruitment of Rad51 to DSB (a key component of HR), homology-directed repair, and survival after DNA damage. Strikingly, p400 and Rad51 are present in the same complex and both favor chromatin remodeling around DSBs. Altogether, our data provide a direct molecular link between Rad51 and a chromatin remodeling enzyme involved in chromatin decompaction around DNA DSBs.

Oxidative Stress-induced DNA Damage in the Synovial Cells of the Temporomandibular Joint in the Rat

2004 ◽  
Vol 83 (8) ◽  
pp. 619-624 ◽  
Author(s):  
T. Yamaza ◽  
K.F. Masuda ◽  
I. Atsuta ◽  
K. Nishijima ◽  
M.A. Kido ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Temporomandibular Joint ◽  
Mechanical Loading ◽  
Enzyme Treatment ◽  
Synovial Cells ◽  
Dna Double Strand Breaks ◽  
Synovial Hyperplasia ◽  
Strand Breaks ◽  
Single Strand Breaks

Synovial hyperplasia is a feature of degenerative temporomandibular joint (TMJ) disease. However, the mechanism by which hyperplasia progresses in the TMJ is unknown. Based on the hypothesis that the oxidative stress generated by mechanical loading causes degenerative changes in the TMJ synovium, we investigated the generation of the highly reactive species, peroxynitrite, and the occurrence of DNA damage in the synovium. After condylar hypermobility of rat TMJs, a marker of peroxynitrite, nitrotyrosine, was localized to the nuclei and cytoplasm of the synovial lining cells and fibroblasts in synovitis-induced TMJ. DNA single-strand breaks were found in the nuclei of the synovial cells only after enzyme treatment, whereas DNA double-strand breaks were not detected. These findings indicate that condylar hypermovement induces the proliferation of synovial cells, and suggest that oxidative stress leads to the progression of synovial hyperplasia via DNA damage of the synovial cells in TMJs after mechanical loading.

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