scholarly journals γ-H2AX Foci Persistence at Chromosome Break Suggests Slow and Faithful Repair Phases Restoring Chromosome Integrity

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1397 ◽  
Author(s):  
Michelle Ricoul ◽  
Tamizh Selvan Gnana Sekaran ◽  
Patricia Brochard ◽  
Cecile Herate ◽  
Laure Sabatier
Keyword(s):  
Dna Damage ◽  
Chromosome Rearrangement ◽  
Chromosome Break ◽  
Dna Double Strand Breaks ◽  
Chromosome Fusion ◽  
Post Irradiation ◽  
Dna Lesion ◽  
Novel Approach ◽  
Chromosome Integrity ◽  
Kinetics Of

Many toxic agents can cause DNA double strand breaks (DSBs), which are in most cases quickly repaired by the cellular machinery. Using ionising radiation, we explored the kinetics of DNA lesion signaling and structural chromosome aberration formation at the intra- and inter-chromosomal level. Using a novel approach, the classic Premature Chromosome Condensation (PCC) was combined with γ-H2AX immunofluorescence staining in order to unravel the kinetics of DNA damage signalisation and chromosome repair. We identified an early mechanism of DNA DSB joining that occurs within the first three hours post-irradiation, when dicentric chromosomes and chromosome exchanges are formed. The slower and significant decrease of ”deleted chromosomes” and 1 acentric telomere fragments observed until 24 h post-irradiation, leads to the conclusion that a second and error-free repair mechanism occurs. In parallel, we revealed remaining signalling of γ-H2AX foci at the site of chromosome fusion long after the chromosome rearrangement formation. Moreover there is important signalling of foci on the site of telomere and sub-telomere sequences suggesting either a different function of γ-H2AX signalling in these regions or an extreme sensibility of the telomere sequences to DNA damage that remains unrepaired 24 h post-irradiation. In conclusion, chromosome repair happens in two steps, including a last and hardly detectable one because of restoration of the chromosome integrity.

scholarly journals PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Deepti Sharma ◽  
Louis De Falco ◽  
Sivaraman Padavattan ◽  
Chang Rao ◽  
Susana Geifman-Shochat ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mutational Analysis ◽  
Catalytic Efficiency ◽  
Double Strand Breaks ◽  
Genomic Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Nucleosome Core ◽  
Epigenetic Marker ◽  
Kinetics Of

AbstractThe poly(ADP-ribose) polymerase, PARP1, plays a key role in maintaining genomic integrity by detecting DNA damage and mediating repair. γH2A.X is the primary histone marker for DNA double-strand breaks and PARP1 localizes to H2A.X-enriched chromatin damage sites, but the basis for this association is not clear. We characterize the kinetics of PARP1 binding to a variety of nucleosomes harbouring DNA double-strand breaks, which reveal that PARP1 associates faster with (γ)H2A.X- versus H2A-nucleosomes, resulting in a higher affinity for the former, which is maximal for γH2A.X-nucleosome that is also the activator eliciting the greatest poly-ADP-ribosylation catalytic efficiency. The enhanced activities with γH2A.X-nucleosome coincide with increased accessibility of the DNA termini resulting from the H2A.X-Ser139 phosphorylation. Indeed, H2A- and (γ)H2A.X-nucleosomes have distinct stability characteristics, which are rationalized by mutational analysis and (γ)H2A.X-nucleosome core crystal structures. This suggests that the γH2A.X epigenetic marker directly facilitates DNA repair by stabilizing PARP1 association and promoting catalysis.

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 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.

Identification of dose-dependent DNA damage and repair responses from subchronic exposure to 1,4-dioxane in mice using a systems analysis approach

2021 ◽  
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

Abstract 1,4-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 Poly(ADP-ribose)-binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions

2020 ◽  
Author(s):  
Ramhari Kumbhar ◽  
Jullian Perren ◽  
Fade Gong ◽  
David Corujo ◽  
Frank Medina ◽  
...  
Keyword(s):  
Dna Damage ◽  
Parp Inhibitors ◽  
Dna Lesions ◽  
Histone Demethylase ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Binding Region ◽  
Transcriptional Responses ◽  
Dna Lesion ◽  
Two Factors

AbstractThe histone demethylase KDM5A removes histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDR). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a non-canonical poly(ADP-ribose), (PAR), binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi) blocks KDM5A-PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and functions at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR-binding and macroH2A1.2 in KDM5A recognition of damage sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.SummaryThe histone demethylase KDM5A demethylates H3K4 to promote repair and transcriptional responses at DNA breaks. We identified poly(ADP-ribose)-binding and macroH2A1.2 as modulators of KDM5A association with DNA damage sites, revealing how KDM5A engages DNA breaks within chromatin.

scholarly journals DNA damage interactions on both nanometer and micrometer scale determine overall cellular damage

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Thomas Friedrich ◽  
Katarina Ilicic ◽  
Christoph Greubel ◽  
Stefanie Girst ◽  
Judith Reindl ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Level ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Cellular Damage ◽  
Biophysical Model ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Lesion ◽  
Micrometer Scale

Abstract DNA double strand breaks (DSB) play a pivotal role for cellular damage, which is a hazard encountered in toxicology and radiation protection, but also exploited e.g. in eradicating tumors in radiation therapy. It is still debated whether and in how far clustering of such DNA lesions leads to an enhanced severity of induced damage. Here we investigate - using focused spots of ionizing radiation as damaging agent - the spatial extension of DNA lesion patterns causing cell inactivation. We find that clustering of DNA damage on both the nm and µm scale leads to enhanced inactivation compared to more homogeneous lesion distributions. A biophysical model interprets these observations in terms of enhanced DSB production and DSB interaction, respectively. We decompose the overall effects quantitatively into contributions from these lesion formation processes, concluding that both processes coexist and need to be considered for determining the resulting damage on the cellular level.

scholarly journals Repair Kinetics of DNA Double Strand Breaks Induced by Simulated Space Radiation

Life ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 341
Author(s):  
Takashi Oizumi ◽  
Rieko Ohno ◽  
Souichiro Yamabe ◽  
Tomoo Funayama ◽  
Asako J. Nakamura
Keyword(s):  
Dna Damage ◽  
Ion Irradiation ◽  
Space Radiation ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Carbon Ion ◽  
Kinetics Of

Radiation is unavoidable in space. Energetic particles in space radiation are reported to induce cluster DNA damage that is difficult to repair. In this study, normal human fibroblasts were irradiated with components of space radiation such as proton, helium, or carbon ion beams. Immunostaining for γ-H2AX and 53BP1 was performed over time to evaluate the kinetics of DNA damage repair. Our data clearly show that the repair kinetics of DNA double strand breaks (DSBs) induced by carbon ion irradiation, which has a high linear energy transfer (LET), are significantly slower than those of proton and helium ion irradiation. Mixed irradiation with carbon ions, followed by helium ions, did not have an additive effect on the DSB repair kinetics. Interestingly, the mean γ-H2AX focus size was shown to increase with LET, suggesting that the delay in repair kinetics was due to damage that is more complex. Further, the 53BP1 focus size also increased in an LET-dependent manner. Repair of DSBs, characterized by large 53BP1 foci, was a slow process within the biphasic kinetics of DSB repair, suggesting non-homologous end joining with error-prone end resection. Our data suggest that the biological effects of space radiation may be significantly influenced by the dose as well as the type of radiation exposure.

scholarly journals Poly(ADP-ribose) binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions

2021 ◽  
Vol 220 (7) ◽  
Author(s):  
Ramhari Kumbhar ◽  
Anthony Sanchez ◽  
Jullian Perren ◽  
Fade Gong ◽  
David Corujo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Parp Inhibitors ◽  
Dna Lesions ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Binding Region ◽  
Homology Directed Repair ◽  
Dna Lesion ◽  
Two Factors ◽  
And Function

The histone demethylase KDM5A erases histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDRs). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a noncanonical poly(ADP-ribose) (PAR)–binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi’s) blocks KDM5A–PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and function at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR binding and macroH2A1.2 in KDM5A recognition of DNA lesion sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.

scholarly journals Making Connections: Integrative Signaling Mechanisms Coordinate DNA Break Repair in Chromatin

2021 ◽  
Vol 12 ◽  
Author(s):  
Anthony Sanchez ◽  
Doohyung Lee ◽  
Dae In Kim ◽  
Kyle M. Miller
Keyword(s):  
Dna Damage ◽  
Protein Interactions ◽  
Dna Lesions ◽  
Genome Integrity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Dsb Repair ◽  
Damage Sensing ◽  
Dna Lesion ◽  
Break Site

DNA double-strand breaks (DSBs) are hazardous to genome integrity and can promote mutations and disease if not handled correctly. Cells respond to these dangers by engaging DNA damage response (DDR) pathways that are able to identify DNA breaks within chromatin leading ultimately to their repair. The recognition and repair of DSBs by the DDR is largely dependent on the ability of DNA damage sensing factors to bind to and interact with nucleic acids, nucleosomes and their modified forms to target these activities to the break site. These contacts orientate and localize factors to lesions within chromatin, allowing signaling and faithful repair of the break to occur. Coordinating these events requires the integration of several signaling and binding events. Studies are revealing an enormously complex array of interactions that contribute to DNA lesion recognition and repair including binding events on DNA, as well as RNA, RNA:DNA hybrids, nucleosomes, histone and non-histone protein post-translational modifications and protein-protein interactions. Here we examine several DDR pathways that highlight and provide prime examples of these emerging concepts. A combination of approaches including genetic, cellular, and structural biology have begun to reveal new insights into the molecular interactions that govern the DDR within chromatin. While many questions remain, a clearer picture has started to emerge for how DNA-templated processes including transcription, replication and DSB repair are coordinated. Multivalent interactions with several biomolecules serve as key signals to recruit and orientate proteins at DNA lesions, which is essential to integrate signaling events and coordinate the DDR within the milieu of the nucleus where competing genome functions take place. Genome architecture, chromatin structure and phase separation have emerged as additional vital regulatory mechanisms that also influence genome integrity pathways including DSB repair. Collectively, recent advancements in the field have not only provided a deeper understanding of these fundamental processes that maintain genome integrity and cellular homeostasis but have also started to identify new strategies to target deficiencies in these pathways that are prevalent in human diseases including cancer.

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

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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