Nucleosomes effectively shield DNA from radiation damage in living cells

Abstract Eukaryotic DNA is organized in nucleosomes, which package DNA and regulate its accessibility to transcription, replication, recombination and repair. Here, we show that in living cells nucleosomes protect DNA from high-energy radiation and reactive oxygen species. We combined sequence-based methods (ATAC-seq and BLISS) to determine the position of both nucleosomes and double strand breaks (DSBs) in the genome of nucleosome-rich malignant mesothelioma cells, and of the same cells partially depleted of nucleosomes. The results were replicated in the human MCF-7 breast carcinoma cell line. We found that, for each genomic sequence, the probability of DSB formation is directly proportional to the fraction of time it is nucleosome-free; DSBs accumulate distal from the nucleosome dyad axis. Nucleosome free regions and promoters of actively transcribed genes are more sensitive to DSB formation, and consequently to mutation. We argue that this may be true for a variety of chemical and physical DNA damaging agents.

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DNA Double-Strand Breaks Induced by High-Energy Neon and Iron Ions in Human Fibroblasts. II. Probing Individual NotI Fragments by Hybridization

Radiation Research ◽

10.2307/3578658 ◽

1994 ◽

Vol 139 (2) ◽

pp. 142 ◽

Cited By ~ 27

Author(s):

Markus Löbrich ◽

Björn Rydberg ◽

Priscilla K. Cooper ◽

Markus Lobrich ◽

Bjorn Rydberg

Keyword(s):

Human Fibroblasts ◽

High Energy ◽

Double Strand Breaks ◽

Iron Ions ◽

Dna Double Strand Breaks ◽

Strand Breaks

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RAD51 localization and activation following DNA damage

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1368 ◽

2004 ◽

Vol 359 (1441) ◽

pp. 87-93 ◽

Cited By ~ 74

Author(s):

Madalena Tarsounas ◽

Adelina A. Davies ◽

Stephen C. West

Keyword(s):

Dna Damage ◽

Genome Stability ◽

Critical Role ◽

S Phase ◽

Double Strand Breaks ◽

Focus Formation ◽

Rad51 Protein ◽

Strand Breaks ◽

Dna Damaging Agents ◽

Binding Level

The efficient repair of double–strand breaks in DNA is critical for the maintenance of genome stability. In response to ionizing radiation and other DNA–damaging agents, the RAD51 protein, which is essential for homologous recombination, relocalizes within the nucleus to form distinct foci that can be visualized by microscopy and are thought to represent sites where repair reactions take place. The formation of RAD51 foci in response to DNA damage is dependent upon BRCA2 and a series of proteins known as the RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), indicating that the components present within foci assemble in a carefully orchestrated and ordered manner. By contrast, RAD51 foci that form spontaneously as cells undergo DNA replication at S phase occur without the need for BRCA2 or the RAD51 paralogues. It is known that BRCA2 interacts directly with RAD51 through a series of degenerative motifs known as the BRC repeats. These interactions modulate the ability of RAD51 to bind DNA. Taken together, these observations indicate that BRCA2 plays a critical role in controlling the actions of RAD51 at both the microscopic (focus formation) and molecular (DNA binding) level.

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Feedback regulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 via ATM/Chk2 pathway contributes to the resistance of MCF-7 breast cancer cells to cisplatin

Tumor Biology ◽

10.1177/1010428317694307 ◽

2017 ◽

Vol 39 (3) ◽

pp. 101042831769430 ◽

Cited By ~ 1

Author(s):

Juan Lv ◽

Ying Qian ◽

Xiaoyan Ni ◽

Xiuping Xu ◽

Xuejun Dong

Keyword(s):

Dna Damage ◽

Cancer Cells ◽

Feedback Regulation ◽

Double Strand Breaks ◽

Gene Clone ◽

Methyl Methanesulfonate ◽

Strand Breaks ◽

Damage Response ◽

Dna Replication And Repair ◽

Mcf 7

The methyl methanesulfonate and ultraviolet-sensitive gene clone 81 protein is a structure-specific nuclease that plays important roles in DNA replication and repair. Knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 has been found to sensitize cancer cells to chemotherapy. However, the underlying molecular mechanism is not well understood. We found that methyl methanesulfonate and ultraviolet-sensitive gene clone 81 was upregulated and the ATM/Chk2 pathway was activated at the same time when MCF-7 cells were treated with cisplatin. By using lentivirus targeting methyl methanesulfonate and ultraviolet-sensitive gene clone 81 gene, we showed that knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 enhanced cell apoptosis and inhibited cell proliferation in MCF-7 cells under cisplatin treatment. Abrogation of ATM/Chk2 pathway inhibited cell viability in MCF-7 cells in response to cisplatin. Importantly, we revealed that ATM/Chk2 was required for the upregulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81, and knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 resulted in inactivation of ATM/Chk2 pathway in response to cisplatin. Meanwhile, knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 activated the p53/Bcl-2 pathway in response to cisplatin. These data suggest that the ATM/Chk2 may promote the repair of DNA damage caused by cisplatin by sustaining methyl methanesulfonate and ultraviolet-sensitive gene clone 81, and the double-strand breaks generated by methyl methanesulfonate and ultraviolet-sensitive gene clone 81 may activate the ATM/Chk2 pathway in turn, which provide a novel mechanism of how methyl methanesulfonate and ultraviolet-sensitive gene clone 81 modulates DNA damage response and repair.

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Homologous Recombination Contributes to the Repair of DNA Double-Strand Breaks Induced by High-Energy Iron Ions

Radiation Research ◽

10.1667/rr1910.1 ◽

2010 ◽

Vol 173 (1) ◽

pp. 27 ◽

Cited By ~ 52

Author(s):

Faria Zafar ◽

Sara B. Seidler ◽

Amy Kronenberg ◽

David Schild ◽

Claudia Wiese

Keyword(s):

Homologous Recombination ◽

High Energy ◽

Double Strand Breaks ◽

Iron Ions ◽

Dna Double Strand Breaks ◽

Strand Breaks

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Cytotoxicity and Genotoxicity Assessment of Sandalwood Essential Oil in Human Breast Cell Lines MCF-7 and MCF-10A

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/3696232 ◽

2016 ◽

Vol 2016 ◽

pp. 1-13 ◽

Cited By ~ 11

Author(s):

Carmen Ortiz ◽

Luisa Morales ◽

Miguel Sastre ◽

William E. Haskins ◽

Jaime Matta

Keyword(s):

Essential Oil ◽

Cell Lines ◽

Double Strand Breaks ◽

Breast Adenocarcinoma ◽

Human Breast ◽

Strand Breaks ◽

Breast Cell Lines ◽

Proteomics Approach ◽

Human Breast Cell ◽

Mcf 7

Sandalwood essential oil (SEO) is extracted fromSantalumtrees. Althoughα-santalol, a main constituent of SEO, has been studied as a chemopreventive agent, the genotoxic activity of the whole oil in human breast cell lines is still unknown. The main objective of this study was to assess the cytotoxic and genotoxic effects of SEO in breast adenocarcinoma (MCF-7) and nontumorigenic breast epithelial (MCF-10A) cells. Proteins associated with SEO genotoxicity were identified using a proteomics approach. Commercially available, high-purity, GC/MS characterized SEO was used to perform the experiments. The main constituents reported in the oil were (Z)-α-santalol (25.34%), (Z)-nuciferol (18.34%), (E)-β-santalol (10.97%), and (E)-nuciferol (10.46%). Upon exposure to SEO (2–8 μg/mL) for 24 hours, cell proliferation was determined by the MTT assay. Alkaline and neutral comet assays were used to assess genotoxicity. SEO exposure induced single- and double-strand breaks selectively in the DNA of MCF-7 cells. Quantitative LC/MS-based proteomics allowed identification of candidate proteins involved in this response: Ku70 (p=1.37E-2), Ku80 (p=5.8E-3), EPHX1 (p=3.3E-3), and 14-3-3ζ(p=4.0E-4). These results provide the first evidence that SEO is genotoxic and capable of inducing DNA single- and double-strand breaks in MCF-7 cells.

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Targeted Inactivation of Mouse RAD52Reduces Homologous Recombination but Not Resistance to Ionizing Radiation

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6423 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6423-6429 ◽

Cited By ~ 213

Author(s):

Tonnie Rijkers ◽

Jody Van Den Ouweland ◽

Bruno Morolli ◽

Anton G. Rolink ◽

Willy M. Baarends ◽

...

Keyword(s):

Homologous Recombination ◽

Es Cells ◽

Embryonic Stem ◽

Double Strand Breaks ◽

Strand Breaks ◽

Dna Damaging Agents ◽

Reduced Frequency ◽

Null Mutant Mice ◽

Targeted Inactivation ◽

Resistance To Ionizing Radiation

ABSTRACT The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant,MmRAD52 −/− ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.

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3H-Thymidine Influence on DNA Double Strand Breaks Induction in Cultured Human Mesenchymal Stem Cells

Medical Radiology and radiation safety ◽

10.12737/article_5a855c9d5b1211.49546901 ◽

2018 ◽

Vol 63 (1) ◽

pp. 28-34 ◽

Cited By ~ 1

Author(s):

Н. Воробьева ◽

N. Vorob'eva ◽

В. Уйба ◽

V. Uyba ◽

О. Кочетков ◽

...

Keyword(s):

Stem Cells ◽

Culture Medium ◽

Specific Radioactivity ◽

Human Mesenchymal Stem Cells ◽

Living Cells ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Immunocytochemical Analysis ◽

Γh2ax Foci

Purpose: To estimate the impact of 3H-thymidine on DNA double strand breaks (DSBs) induction in cultured human mesenchymal stem cells (MSC). Material and methods: Isolation and cultivation of human bone marrow MSC was carried out according to a standard procedure. A sterile solution of 3H-thymidine with different specific radioactivity was added to the cell culture and incubated under the conditions of the CO2 incubator for 24 hours. The specific radioactivity of 3H-thymidine in the incubation medium was 50–1600 kBq/ml. To evaluate quantitatively the DSBs, an immunocytochemical analysis of the DSB marker – γH2AX foci histone was used. Additionally, the proportion of dividing cells was estimated using an immunocytochemical analysis of the cell proliferation marker, the Ki67 protein. Results: It was shown that 24 h incubation of human MSC in a culture medium results in a dose-dependent increase in γH2AX foci. There is a linear increase in the foci γH2AX in the range of 50–400 kBq/ml, after which the relative quantitative yield of foci per unit of specific radioactivity begins to decrease. In general, the dose-effect relationship is approximated by the quadratic function y = 3.13 + 50.80x – 12.38x2 (R2 = 0.99), where y is the number of foci γH2AX in the cell nucleus, and x is the specific radioactivity in 1000 kBq/ml. It was found that incubation of human MSC in a culture medium containing 800 and 1600 kBq/ml of 3H-thymidine resulted in a statistically significant decrease in the cells proliferative activity compared to the control of ~1.25 and 1.41 respectively. The peculiar biological limitation of tritium accumulation in the cell nucleus explains well the nonlinear character of the dependence of the formation of DSBs on the specific radioactivity of 3H-thymidine in the culture medium observed in our study. Conclusion: Quantitative analysis of γH2AX foci has proved to be a highly reproducible and highly sensitive method for evaluating the induction of DSBs in living cells under the action of 3H-thymidine. An analysis of the foci of γH2AX will be useful for accurate estimating the quantitative yield of DBS in living cells per dose of 3H-thymidine β-radiation. To do this, it is necessary to make a correct calculation of the doses received by the cells taking into account the microdistribution of 3H-thymidine in the cell volume and its accumulation in the DNA of living cells.

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Requirement for End-Joining and Checkpoint Functions, but NotRAD52-Mediated Recombination, after EcoRI Endonuclease Cleavage of Saccharomyces cerevisiaeDNA

Molecular and Cellular Biology ◽

10.1128/mcb.18.4.1891 ◽

1998 ◽

Vol 18 (4) ◽

pp. 1891-1902 ◽

Cited By ~ 46

Author(s):

L. Kevin Lewis ◽

Jakob M. Kirchner ◽

Michael A. Resnick

Keyword(s):

Nuclear Dna ◽

Double Strand Breaks ◽

Yeast Cells ◽

Chromosomal Dna ◽

End Joining ◽

Strand Breaks ◽

Dna Damaging Agents ◽

Physical And Chemical ◽

Type Strains

ABSTRACT RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired inrad52 mutants and in G1-phase Rad+cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly,rad52 mutants were not more sensitive toEcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly whenEcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.

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Single molecule microscopy reveals key physical features of repair foci in living cells

eLife ◽

10.7554/elife.60577 ◽

2021 ◽

Vol 10 ◽

Author(s):

Judith Miné-Hattab ◽

Mathias Heltberg ◽

Marie Villemeur ◽

Chloé Guedj ◽

Thierry Mora ◽

...

Keyword(s):

Single Molecule ◽

Liquid Droplet ◽

Physical Nature ◽

Living Cells ◽

Double Strand Breaks ◽

Single Molecule Microscopy ◽

Dsb Repair ◽

Strand Breaks ◽

Functional Homolog ◽

Physical Features

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

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Illegitimate Recombination Induced by Overproduction of DnaB Helicase in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.181.15.4549-4553.1999 ◽

1999 ◽

Vol 181 (15) ◽

pp. 4549-4553 ◽

Cited By ~ 10

Author(s):

Teruhito Yamashita ◽

Katsuhiro Hanada ◽

Mihoko Iwasaki ◽

Hirotaka Yamaguchi ◽

Hideo Ikeda

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Low Frequency ◽

Double Strand Breaks ◽

Illegitimate Recombination ◽

Replication Forks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dna Damaging Agents ◽

Dnab Helicase

ABSTRACT Illegitimate recombination that usually takes place at a low frequency is greatly enhanced by treatment with DNA-damaging agents. It is thought that DNA double-strand breaks induced by this DNA damage are important for initiation of illegitimate recombination. Here we show that illegitimate recombination is enhanced by overexpression of the DnaB protein in Escherichia coli. The recombination enhanced by DnaB overexpression occurred between short regions of homology. We propose a model for the initiation of illegitimate recombination in which DnaB overexpression may excessively unwind DNA at replication forks and induce double-strand breaks, resulting in illegitimate recombination. The defect in RecQ has a synergistic effect on the increased illegitimate recombination in cells containing the overproduced DnaB protein, implying that DnaB works in the same pathway as RecQ does but that they work at different steps.

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