scholarly journals Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells

2020 ◽  
Vol 21 (18) ◽  
pp. 6602 ◽  
Author(s):  
Stefan J. Roobol ◽  
Irene van den Bent ◽  
Wiggert A. van Cappellen ◽  
Tsion E. Abraham ◽  
Maarten W. Paul ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein A ◽  
Strand Break ◽  
Double Strand Break ◽  
Protein Accumulation ◽  
Dna Double Strand Breaks ◽  
X Ray ◽  
High Let ◽  
Dna End Resection ◽  
Α Particles

High-linear-energy-transfer (LET) radiation is more lethal than similar doses of low-LET radiation types, probably a result of the condensed energy deposition pattern of high-LET radiation. Here, we compare high-LET α-particle to low-LET X-ray irradiation and monitor double-strand break (DSB) processing. Live-cell microscopy was used to monitor DNA double-strand breaks (DSBs), marked by p53-binding protein 1 (53BP1). In addition, the accumulation of the endogenous 53BP1 and replication protein A (RPA) DSB processing proteins was analyzed by immunofluorescence. In contrast to α-particle-induced 53BP1 foci, X-ray-induced foci were resolved quickly and more dynamically as they showed an increase in 53BP1 protein accumulation and size. In addition, the number of individual 53BP1 and RPA foci was higher after X-ray irradiation, while focus intensity was higher after α-particle irradiation. Interestingly, 53BP1 foci induced by α-particles contained multiple RPA foci, suggesting multiple individual resection events, which was not observed after X-ray irradiation. We conclude that high-LET α-particles cause closely interspaced DSBs leading to high local concentrations of repair proteins. Our results point toward a change in DNA damage processing toward DNA end-resection and homologous recombination, possibly due to the depletion of soluble protein in the nucleoplasm. The combination of closely interspaced DSBs and perturbed DNA damage processing could be an explanation for the increased relative biological effectiveness (RBE) of high-LET α-particles compared to X-ray irradiation.

scholarly journals SIRT6 is a DNA Double-Strand Break Sensor

2019 ◽  
Author(s):  
Lior Onn ◽  
Miguel Portillo ◽  
Stefan Ilic ◽  
Gal Cleitman ◽  
Daniel Stein ◽  
...  
Keyword(s):  
Dna Damage ◽  
Binding Capacity ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
The Road ◽  
Non Homologous End Joining

AbstractDNA double strand breaks are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure, with high affinity for double strand breaks. It relocates to sites of damage independently of signalling and known sensors and activates downstream signalling cascades for double strand break repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the Homologous Recombination and Non-Homologous End Joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as DNA damage sensor, which is critical for initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB binding capacity and DDR activation. SIRT6 activates the DDR, before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 at the top of the DDR and pave the road to dissect the contributions of distinct double strand break sensors in downstream signalling.

scholarly journals Metal Ions Modify in Vitro DNA Damage Yields With High-LET Radiation

2021 ◽  
Author(s):  
Dylan Buglewicz ◽  
Cathy Su ◽  
Austin Banks ◽  
Jazmine Strenger-smith ◽  
Suad Elmegerhi ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Strand Break ◽  
Single Strand ◽  
Iron Ion ◽  
Single Strand Break ◽  
Carbon Ion ◽  
X Ray ◽  
High Let ◽  
Break Formation

Abstract Cu2+ and Co2+ are metals known to increase DNA damage in the presence of hydrogen peroxide through a Fenton type reaction. We hypothesized that these metals could increase DNA damage following irradiations of increasing LET values as hydrogen peroxide is a product of the radiolysis of water. The reaction mixtures contain either double- or single-stranded DNA in solution with Cu2+ or Co2+ and was irradiated either with X-ray, carbon-ion or iron-ion beams or was treated with hydrogen peroxide or bleomycin at increasing radiation dosages or chemical concentrations. DNA damage was then assessed by gel electrophoresis followed by band intensity analysis. DNA in solution with metals demonstrated the most DNA damage when treated with hydrogen peroxide followed by irradiation with low-LET (X-Ray), high-LET (carbon-ion and iron-ion), respectively, and demonstrated the least damage with treatment of bleomycin. Cu2+ portrayed greater DNA damage than Co2+ following all experimental conditions. The metals effect caused more DNA damage and was observed to be LET dependent for single-strand break formation but inversely dependent for double-strand break formation. These results suggest that Cu2+ is more efficient than Co2+ at inducing both DNA single-strand and double-strand breaks following all irradiations and chemical treatments.

scholarly journals The Effect of Atypical Nucleic Acids Structures in DNA Double Strand Break Repair: A Tale of R-loops and G-Quadruplexes

2021 ◽  
Vol 12 ◽  
Author(s):  
Rosa Camarillo ◽  
Sonia Jimeno ◽  
Pablo Huertas
Keyword(s):  
Nucleic Acids ◽  
Strand Break ◽  
Double Strand Break ◽  
Fine Tuning ◽  
Double Strand Break Repair ◽  
Dna Double Strand Breaks ◽  
Dna Double Strand Break ◽  
Break Repair ◽  
Dna End Resection ◽  
End Resection

The fine tuning of the DNA double strand break repair pathway choice relies on different regulatory layers that respond to environmental and local cues. Among them, the presence of non-canonical nucleic acids structures seems to create challenges for the repair of nearby DNA double strand breaks. In this review, we focus on the recently published effects of G-quadruplexes and R-loops on DNA end resection and homologous recombination. Finally, we hypothesized a connection between those two atypical DNA structures in inhibiting the DNA end resection step of HR.

scholarly journals Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage

2005 ◽  
Vol 391 (3) ◽  
pp. 473-480 ◽  
Author(s):  
Xiaoming Wu ◽  
Zhengguan Yang ◽  
Yiyong Liu ◽  
Yue Zou
Keyword(s):  
Dna Damage ◽  
Protein A ◽  
Replication Protein A ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Protein ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Checkpoint Activation ◽  
In Cells

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

scholarly journals Double Stranded DNA Breaks and Genome Editing Trigger Ribosome Remodeling and Translational Shutdown

2018 ◽  
Author(s):  
Celeste Riepe ◽  
Elena Zelin ◽  
Stacia K. Wyman ◽  
David N. Nguyen ◽  
Jin Rui Liang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Editing ◽  
Ribosomal Proteins ◽  
Transcriptional Response ◽  
Strand Break ◽  
Double Strand Break ◽  
Initiation Factor ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Cellular Phenotypes

SummaryDNA damage activates a robust transcriptional stress response, but much less is known about how DNA impacts translation. The advent of genome editing via a Cas9-induced DNA double-strand break has intensified interest in understanding cellular responses to DNA damage. Here we find that DNA double-strand breaks (DSBs) induced by Cas9 or other damaging agents lead to a reduction of core ribosomal proteins, RPS27A and RPL40, and that the loss of these proteins is post-transcriptional and p53-independent. DSBs furthermore lead to the shutdown of translation through phosphorylation of eukaryotic initiation factor 2 alpha, and altering these signals affects genome editing outcomes. This DSB translational response is widespread and precedes the transcriptional response. Our results demonstrate that even a single double-strand break can lead to ribosome remodeling and reduced translational output, and suggest caution in interpreting cellular phenotypes measured immediately after genome editing.

scholarly journals Evaluating Gamma-H2AX Expression as a Biomarker of DNA Damage after X-ray in Angiography Patients

2018 ◽  
Vol 8 (4Dec) ◽  
Author(s):  
A Alipoor ◽  
R Fardid ◽  
S Sharifzadeh
Keyword(s):  
Dna Damage ◽  
Biological Effects ◽  
Strand Break ◽  
Double Strand Break ◽  
Peripheral Blood Lymphocytes ◽  
Dna Double Strand Breaks ◽  
Histone H2ax ◽  
Blood Lymphocytes ◽  
Dna Double Strand Break ◽  
Effects Of Radiation

Objective: Coronary heart disease (CHD) is one of the most common diseases. Coronary angiography (CAG) is an important apparatus used to diagnose and treat this disease. Since angiography is performed through exposure to ionizing radiation, it can cause harmful effects induced by double-stranded breaks in DNA which is potentially life-threatening damage. The aim of the present study is to investigate phosphorylation of Histone H2AX in the location of double-stranded breaks in peripheral blood lymphocytes as an indication of biological effects of radiation on angiography.Materials and Methods: This method is based on the phosphorylation measurement of Histone (gamma-H2AX or γ-H2AX) levels on serine 139 after the formation of DNA double-strand break. 5 cc of blood samples from 24 patients undergoing angiography were taken pre- and post-radiation. Blood lymphocytes were extracted, fixed and stained with specific γ-H2AX antibodies. Finally, the percentage of phosphorylation of Histone H2AX as an indicator of double-strand break was measured by a cytometry technique.Results: An increase was observed in all patients’ percentage of phosphorylated Histone H2AX (double-stranded breaks DNA) after radiation (20.15 ± 14.18) compared to pre-exposure time (1.52 ± 0.34). Also, the mean of DNA double-strand break is shown in a linear correlation with DAP.Discussion: Although induction of DNA double-strand breaks was associated with the radiation dose in patients, the effect of individual factors such as radio-sensitivity and regenerative capacity should not be ignored. In the future, if we are able to measure DNA damage response in every angiography patient, we will use it as a biomarker for the patient dose; this will promote public health.Conclusion: Using flow cytometers readings done automatically is possible to detect γ-H2AX in the number of blood cells, therefore, the use of this technique could play a significant role in monitoring patients.

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.

scholarly journals DNA structure-specific priming of ATR activation by DNA-PKcs

2013 ◽  
Vol 202 (3) ◽  
pp. 421-429 ◽  
Author(s):  
Sophie Vidal-Eychenié ◽  
Chantal Décaillet ◽  
Jihane Basbous ◽  
Angelos Constantinou
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Protein A ◽  
Dna Structure ◽  
Specific Response ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Synergistic Activation ◽  
Dependent Protein ◽  
Specific Priming

Three phosphatidylinositol-3-kinase–related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)–covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.

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