DNA damage alters nuclear mechanics through chromatin reorganization

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.

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DNA damage alters nuclear mechanics through chromatin reorganisation

10.1101/2020.07.10.197517 ◽

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.

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Actin-Related Protein 6 (Arp6) Influences Double-Strand Break Repair in Yeast

Applied Microbiology ◽

10.3390/applmicrobiol1020017 ◽

2021 ◽

Vol 1 (2) ◽

pp. 225-238

Author(s):

Mohsen Hooshyar ◽

Daniel Burnside ◽

Maryam Hajikarimlou ◽

Katayoun Omidi ◽

Alexander Jesso ◽

...

Keyword(s):

Dna Damage ◽

Chromatin Remodeling ◽

Genetic Interaction ◽

Interaction Analysis ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Dna Damaging Agents ◽

Repair Efficiency ◽

Chromosomal Breaks

DNA double-strand breaks (DSBs) are the most deleterious form of DNA damage and are repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR). Repair initiation, regulation and communication with signaling pathways require several histone-modifying and chromatin-remodeling complexes. In budding yeast, this involves three primary complexes: INO80-C, which is primarily associated with HR, SWR1-C, which promotes NHEJ, and RSC-C, which is involved in both pathways as well as the general DNA damage response. Here we identify ARP6 as a factor involved in DSB repair through an RSC-C-related pathway. The loss of ARP6 significantly reduces the NHEJ repair efficiency of linearized plasmids with cohesive ends, impairs the repair of chromosomal breaks, and sensitizes cells to DNA-damaging agents. Genetic interaction analysis indicates that ARP6, MRE11 and RSC-C function within the same pathway, and the overexpression of ARP6 rescues rsc2∆ and mre11∆ sensitivity to DNA-damaging agents. Double mutants of ARP6, and members of the INO80 and SWR1 complexes, cause a significant reduction in repair efficiency, suggesting that ARP6 functions independently of SWR1-C and INO80-C. These findings support a novel role for ARP6 in DSB repair that is independent of the SWR1 chromatin remodeling complex, through an apparent RSC-C and MRE11-associated DNA repair pathway.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906102116 ◽

2019 ◽

Vol 116 (39) ◽

pp. 19552-19562 ◽

Cited By ~ 7

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.

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Variability in DNA repair and individual susceptibility to genotoxins

Clinical Chemistry ◽

10.1093/clinchem/41.12.1848 ◽

1995 ◽

Vol 41 (12) ◽

pp. 1848-1853 ◽

Cited By ~ 16

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.

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

Nature Communications ◽

10.1038/s41467-019-12929-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 2

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.

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

Blood ◽

10.1182/blood-2007-05-092510 ◽

2008 ◽

Vol 111 (6) ◽

pp. 3173-3182 ◽

Cited By ~ 165

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.

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

10.1101/2020.12.27.424470 ◽

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.

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Assessment of DNA damage and repair efficiency in drug naïve schizophrenia using comet assay

Journal of Psychiatric Research ◽

10.1016/j.jpsychires.2015.05.018 ◽

2015 ◽

Vol 68 ◽

pp. 47-53 ◽

Cited By ~ 9

Author(s):

Aparna Muraleedharan ◽

Vikas Menon ◽

Ravi Philip Rajkumar ◽

Parkash Chand

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Dna Damage And Repair ◽

Repair Efficiency ◽

Drug Naïve

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BCR/ABL Expression Increases the Formation of Chromosomal Translocations after DNA Damage.

Blood ◽

10.1182/blood.v104.11.713.713 ◽

2004 ◽

Vol 104 (11) ◽

pp. 713-713 ◽

Cited By ~ 1

Author(s):

Jamil Dierov ◽

Hesed Padilla-Nash ◽

Thomas Ried ◽

Martin Carroll

Keyword(s):

Dna Damage ◽

Genomic Instability ◽

Cellular Response ◽

Blast Crisis ◽

Chronic Phase ◽

Annexin V ◽

Genotoxic Stress ◽

Protein Product ◽

Chromosomal Translocations ◽

Dna Double Strand Breaks

Abstract BCR/ABL is the protein product of the t(9;22) translocation and is the cause of the hyperproliferation associated with chronic phase chronic myeloid leukemia (CML). However, whether BCR/ABL induces genomic instability leading to blast crisis is controversial. We have previously demonstrated that BCR/ABL translocates to the nucleus after genotoxic damage and associates with the DNA damage sensor, ataxia telangiectasia and rad 3 related (ATR) protein, disrupting the sensing and repair of DNA double strand breaks. Here, we have asked if BCR/ABL expression leads to permanent changes in the DNA after genotoxic stress. For these experiments we have studied the hematopoietic cell line, Ba/F3pTetOn p210, which expresses p210 BCR/ABL after incubation in doxycycline. Cells were incubated in low doses of etoposide for two hours and then allowed to recover for 48 hours in the absence of further DNA damage. Induction of apoptosis in these conditions was consistently less than 5% as demonstrated by annexin V staining of cells. Cells were examined for alterations in the chromosomes using Giemsa banding and spectral karyotyping (SKY). Cells growing in IL3 showed low levels of DNA damage with a few broken chromosomes present in metaphase spreads and an average of 0.5 new chromosomal translocations per cell as revealed by SKY analysis. In contrast, when cells expressing BCR/ABL were treated with the same conditions, a marked number of genetic abnormalities were seen. 75% of cells showed abnormal chromosome forms with ring chromosomes, triradial forms and other abnormalitites. Analysis of BCR/ABL expressing cells by SKY analysis showed frequent abnormalities, averaging at least 6 new translocations per cell. Several cells had greater than 10 translocations present including multiple complex translocations involving more than two chromosomes. The majority of abnormalities consisted of unbalanced translocations. This data demonstrates that BCR/ABL alters the cellular response to DNA damage leading to an increase in chromosomal translocations in cells expressing the oncogene and suggests that BCR/ABL itself is directly responsible for the genomic instability leading to CML blast crisis.

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