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 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 Particles with similar LET values generate DNA breaks of different complexity and reparability: a high-resolution microscopy analysis of γH2AX/53BP1 foci

Nanoscale ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 1162-1179 ◽  
Author(s):  
Lucie Jezkova ◽  
Mariia Zadneprianetc ◽  
Elena Kulikova ◽  
Elena Smirnova ◽  
Tatiana Bulanova ◽  
...  
Keyword(s):  
Dna Damage ◽  
High Resolution ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Dna Breaks ◽  
Dna Double Strand Break ◽  
Different Types ◽  
Microscopy Analysis ◽  
High Resolution Microscopy

Different particles with similar LET and energy may generate different types of DNA damage with consequences for DNA double-strand break repair.

scholarly journals YGR042W/MTE1 Functions in Double-Strand Break Repair with MPH1

2015 ◽  
Author(s):  
Askar Yimit ◽  
TaeHyung Kim ◽  
Ranjith Anand ◽  
Sarah Meister ◽  
Jiongwen Ou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Helicase ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Break Repair

Double-strand DNA breaks occur upon exposure of cells to agents such as ionizing radiation and ultraviolet light or indirectly through replication fork collapse at DNA damage sites. If left unrepaired double-strand breaks can cause genome instability and cell death. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination re-localize into discrete nuclear foci. We identified 29 proteins that co-localize with the recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase Mph1 is absent. Mte1 and Mph1 form a complex, and are recruited to double-strand breaks in vivo in a mutually dependent manner. Mte1 is important for resolution of Rad52 foci during double-strand break repair, and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.

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 The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

2019 ◽  
Vol 47 (6) ◽  
pp. 2703-2715 ◽  
Author(s):  
Naike Salvador Moreno ◽  
Jing Liu ◽  
Karen M Haas ◽  
Laurie L Parker ◽  
Chaitali Chakraborty ◽  
...  
Keyword(s):  
Dna Damage ◽  
Parp Inhibitor ◽  
Strand Break ◽  
Negative Regulator ◽  
Structural Protein ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Additional Level

Abstract P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

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 ATM-dependent pathways of chromatin remodelling and oxidative DNA damage responses

2017 ◽  
Vol 372 (1731) ◽  
pp. 20160283 ◽  
Author(s):  
N. Daniel Berger ◽  
Fintan K. T. Stanley ◽  
Shaun Moore ◽  
Aaron A. Goodarzi
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Strand Break ◽  
Chromatin Remodelling ◽  
Double Strand Breaks ◽  
Biochemical Network ◽  
Regulatory Function ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
The Impact

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase with a master regulatory function in the DNA damage response. In this role, ATM commands a complex biochemical network that signals the presence of oxidative DNA damage, including the dangerous DNA double-strand break, and facilitates subsequent repair. Here, we review the current state of knowledge regarding ATM-dependent chromatin remodelling and epigenomic alterations that are required to maintain genomic integrity in the presence of DNA double-strand breaks and/or oxidative stress. We will focus particularly on the roles of ATM in adjusting nucleosome spacing at sites of unresolved DNA double-strand breaks within complex chromatin environments, and the impact of ATM on preserving the health of cells within the mammalian central nervous system. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

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