scholarly journals DNA damage alters nuclear mechanics through chromatin reorganisation

2020 ◽  
Author(s):  
Ália dos Santos ◽  
Alexander W. Cook ◽  
Rosemarie E Gough ◽  
Martin Schilling ◽  
Nora Aleida Olszok ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genomic Instability ◽  
Molecular Diffusion ◽  
Biomechanical Properties ◽  
Protective Role ◽  
Dna Lesions ◽  
Dna Double Strand Breaks ◽  
Dna Damage And Repair ◽  
Nuclear Mechanics ◽  
Dna Damage Signalling

ABSTRACTDNA double-strand breaks (DSBs) drive genomic instability. For efficient and accurate repair of these DNA lesions, the cell activates DNA damage repair pathways. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency.Here, we used Atomic Force Microscopy (AFM) to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

scholarly journals DNA damage alters nuclear mechanics through chromatin reorganization

2020 ◽  
Author(s):  
Ália dos Santos ◽  
Alexander W Cook ◽  
Rosemarie E Gough ◽  
Martin Schilling ◽  
Nora A Olszok ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genomic Instability ◽  
Molecular Diffusion ◽  
Biomechanical Properties ◽  
Protective Role ◽  
Dna Double Strand Breaks ◽  
Dna Damage And Repair ◽  
Nuclear Mechanics ◽  
Repair Efficiency ◽  
Dna Damage Signalling

Abstract DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

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 Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response

2019 ◽  
Vol 116 (39) ◽  
pp. 19552-19562 ◽  
Author(s):  
Justine Sitz ◽  
Sophie Anne Blanchet ◽  
Steven F. Gameiro ◽  
Elise Biquand ◽  
Tia M. Morgan ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Genomic Instability ◽  
E3 Ligase ◽  
Human Papillomaviruses ◽  
Double Strand Breaks ◽  
Nuclear Bodies ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell’s DNA replication and repair machineries to replicate their own genomes. How this host–pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

Variability in DNA repair and individual susceptibility to genotoxins

1995 ◽  
Vol 41 (12) ◽  
pp. 1848-1853 ◽  
Author(s):  
S A Kyrtopoulos
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protective Role ◽  
Interindividual Variation ◽  
Human Populations ◽  
Cytostatic Drugs ◽  
Dna Damage And Repair ◽  
Cellular Protection ◽  
Methylated Dna ◽  
Methylating Agents

Abstract DNA repair is an important mechanism of cellular protection from the effects of genotoxic chemicals. Although extensive evidence from studies in experimental systems indicates that variation in DNA repair can significantly influence susceptibility to genotoxins, corresponding studies in human populations are so far limited, mainly because of methodological difficulties. One system, using observations of the accumulation and repair of DNA damage in cancer patients treated with alkylating cytostatic drugs, has provided useful information for assessing the effects of interindividual variation in DNA repair activity on the induction of genotoxic effects in humans. The most detailed studies of this kind have been carried out on patients with cancer (i.e., Hodgkin disease, malignant melanoma) treated with the methylating cytostatic drugs procarbazine or dacarbazine; these studies have provided detailed information on dose-response relationships. They have also demonstrated the protective role of the repair enzyme O6-alkylguanine-DNA alkyltransferase against the accumulation of the premutagenic methylated DNA lesion O6-methylguanine in patients' DNA. Given the strong evidence that exposure of the general population to environmental methylating agents may be extensive, as indicated by the frequent discovery of methylated DNA adducts in human DNA, data on DNA damage and repair in alkylating drug-treated patients and their modulation by host factors may prove useful in efforts to assess the possible carcinogenic risks posed by exposure to environmental methylating agents.

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 MDC1 PST-repeat region promotes histone H2AX-independent chromatin association and DNA damage tolerance

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Israel Salguero ◽  
Rimma Belotserkovskaya ◽  
Julia Coates ◽  
Matylda Sczaniecka-Clift ◽  
Mukerrem Demir ◽  
...  
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Repeat Region ◽  
Independent Function ◽  
Dna Double Strand Breaks ◽  
Dna Damage Tolerance ◽  
Strand Breaks ◽  
Histone H2ax ◽  
Independent Association ◽  
Dna Damage Signalling

AbstractHistone H2AX and MDC1 are key DNA repair and DNA-damage signalling proteins. When DNA double-strand breaks (DSBs) occur, H2AX is phosphorylated and then recruits MDC1, which in turn serves as a docking platform to promote the localization of other factors, including 53BP1, to DSB sites. Here, by using CRISPR-Cas9 engineered human cell lines, we identify a hitherto unknown, H2AX-independent, function of MDC1 mediated by its PST-repeat region. We show that the PST-repeat region directly interacts with chromatin via the nucleosome acidic patch and mediates DNA damage-independent association of MDC1 with chromatin. We find that this region is largely functionally dispensable when the canonical γH2AX-MDC1 pathway is operative but becomes critical for 53BP1 recruitment to DNA-damage sites and cell survival following DSB induction when H2AX is not available. Consequently, our results suggest a role for MDC1 in activating the DDR in areas of the genome lacking or depleted of H2AX.

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.

Internal tandem duplication of FLT3 (FLT3/ITD) induces increased ROS production, DNA damage, and misrepair: implications for poor prognosis in AML

Blood ◽  
2008 ◽  
Vol 111 (6) ◽  
pp. 3173-3182 ◽  
Author(s):  
Annahita Sallmyr ◽  
Jinshui Fan ◽  
Kamal Datta ◽  
Kyu-Tae Kim ◽  
Dan Grosu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genomic Instability ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Tandem Duplication ◽  
Ros Generation ◽  
Ros Production ◽  
Dna Double Strand Breaks ◽  
Internal Tandem Duplication ◽  
Downstream Signaling

Abstract Activating mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor occur in approximately 30% of acute myeloid leukemia (AML) patients and, at least for internal tandem duplication (ITD) mutations, are associated with poor prognosis. FLT3 mutations trigger downstream signaling pathways including RAS-MAP/AKT kinases and signal transducer and activator of transcription-5 (STAT5). We find that FLT3/ITD mutations start a cycle of genomic instability whereby increased reactive oxygen species (ROS) production leads to increased DNA double-strand breaks (DSBs) and repair errors that may explain aggressive AML in FLT3/ITD patients. Cell lines transfected with FLT3/ITD and FLT3/ITD-positive AML cell lines and primary cells demonstrate increased ROS. Increased ROS levels appear to be produced via STAT5 signaling and activation of RAC1, an essential component of ROS-producing NADPH oxidases. A direct association of RAC1-GTP binding to phosphorylated STAT5 (pSTAT5) provides a possible mechanism for ROS generation. A FLT3 inhibitor blocked increased ROS in FLT3/ITD cells resulting in decreased DSB and increased repair efficiency and fidelity. Our study suggests that the aggressiveness of the disease and poor prognosis of AML patients with FLT3/ITD mutations could be the result of increased genomic instability that is driven by higher endogenous ROS, increased DNA damage, and decreased end-joining fidelity.

scholarly journals Novel insights into the mode of action of 1,4-dioxane using a systems screening approach

2020 ◽  
Author(s):  
Georgia Charkoftaki ◽  
Jaya Prakash Golla ◽  
Alvaro Santos-Neto ◽  
David J. Orlicky ◽  
Rolando Garcia-Milian ◽  
...  
Keyword(s):  
Dna Damage ◽  
Drinking Water ◽  
Immunohistochemical Analysis ◽  
The United States ◽  
Environmental Chemicals ◽  
Dna Double Strand Breaks ◽  
Dna Damage And Repair ◽  
Mechanisms Of Toxicity ◽  
Novel Approach ◽  
Histopathological Studies

Abstract1,4-Dioxane (1,4-DX) is an environmental contaminant found in drinking water throughout the United States (US). While it is a suspected liver carcinogen, there is no federal or state maximum contaminant level for 1,4-DX in drinking water. Very little is known about the mechanisms by which this chemical elicits liver carcinogenicity. In the present study, female BDF-1 mice were exposed to 1,4-DX (0, 50, 500 and 5,000 mg/L) in their drinking water for one or four weeks, to explore the toxic effects. Histopathological studies and a multi-omics approach (transcriptomics and metabolomics) were performed to investigate potential mechanisms of toxicity. Immunohistochemical analysis of the liver revealed increased H2AXγ-positive hepatocytes (a marker of DNA double strand breaks), and an expansion of precholangiocytes (reflecting both DNA damage and repair mechanisms) after exposure. Liver transcriptomics revealed 1,4-DX-induced perturbations in signaling pathways predicted to impact the oxidative stress response, detoxification, and DNA damage. Liver, kidney, feces and urine metabolomic profiling revealed no effect of 1,4-DX exposure, and bile acid quantification in liver and feces similarly showed no effect of exposure. We speculate that the results may be reflective of DNA damage being counterbalanced by the repair response, with the net result being a null overall effect on the systemic biochemistry of the exposed mice. Our results show a novel approach for the investigation of environmental chemicals that do not elicit cell death but have activated the repair systems in response to 1,4-DX exposure.

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.

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