Detection of genomic DNA damage induced by genotoxins in mammalian cells by a chemiluminescence micro-plate DNA repair assay

1998 ◽  
Vol 95 ◽  
pp. 40
Author(s):  
B. Salles ◽  
R.-Y. Li ◽  
C. Bozzato
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Genomic Dna ◽  
Mammalian Cells

scholarly journals Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage

2008 ◽  
Vol 105 (46) ◽  
pp. 17801-17806 ◽  
Author(s):  
Giovanna Pontarin ◽  
Artur Fijolek ◽  
Paola Pizzo ◽  
Paola Ferraro ◽  
Chiara Rampazzo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Mitochondrial Dna ◽  
Dna Replication ◽  
Ribonucleotide Reductase ◽  
Mammalian Cells ◽  
S Phase ◽  
Mt Dna ◽  
Intact Cells ◽  
Mitochondrial Dna Depletion

Ribonucleotide reductase provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and repair. The mammalian enzyme consists of a catalytic (R1) and a radical-generating (R2 or p53R2) subunit. During S-phase, a R1/R2 complex is the major provider of deoxynucleotides. p53R2 is induced by p53 after DNA damage and was proposed to supply deoxynucleotides for DNA repair after translocating from the cytosol to the cell nucleus. Similarly R1 and R2 were claimed to move to the nucleus during S-phase to provide deoxynucleotides for DNA replication. These models suggest translocation of ribonucleotide reductase subunits as a regulatory mechanism. In quiescent cells that are devoid of R2, R1/p53R2 synthesizes deoxynucleotides also in the absence of DNA damage. Mutations in human p53R2 cause severe mitochondrial DNA depletion demonstrating a vital function for p53R2 different from DNA repair and cast doubt on a nuclear localization of the protein. Here we use three independent methods to localize R1, R2, and p53R2 in fibroblasts during cell proliferation and after DNA damage: Western blotting after separation of cytosol and nuclei; immunofluorescence in intact cells; and transfection with proteins carrying fluorescent tags. We thoroughly validate each method, especially the specificity of antibodies. We find in all cases that ribonucleotide reductase resides in the cytosol suggesting that the deoxynucleotides produced by the enzyme diffuse into the nucleus or are transported into mitochondria and supporting a primary function of p53R2 for mitochondrial DNA replication.

scholarly journals DNA Repair Pathways in Cancer Therapy and Resistance

2021 ◽  
Vol 11 ◽  
Author(s):  
Lan-ya Li ◽  
Yi-di Guan ◽  
Xi-sha Chen ◽  
Jin-ming Yang ◽  
Yan Cheng
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Mammalian Cells ◽  
Chemotherapeutic Agents ◽  
Cancer Therapies ◽  
Exogenous Dna ◽  
Dna Damaging Agents ◽  
Anti Cancer ◽  
Mitochondria Dna ◽  
Repair Pathways

DNA repair pathways are triggered to maintain genetic stability and integrity when mammalian cells are exposed to endogenous or exogenous DNA-damaging agents. The deregulation of DNA repair pathways is associated with the initiation and progression of cancer. As the primary anti-cancer therapies, ionizing radiation and chemotherapeutic agents induce cell death by directly or indirectly causing DNA damage, dysregulation of the DNA damage response may contribute to hypersensitivity or resistance of cancer cells to genotoxic agents and targeting DNA repair pathway can increase the tumor sensitivity to cancer therapies. Therefore, targeting DNA repair pathways may be a potential therapeutic approach for cancer treatment. A better understanding of the biology and the regulatory mechanisms of DNA repair pathways has the potential to facilitate the development of inhibitors of nuclear and mitochondria DNA repair pathways for enhancing anticancer effect of DNA damage-based therapy.

scholarly journals A CRISPR-associated factor Csa3a regulates DNA damage repair in Crenarchaeon Sulfolobus islandicus

2020 ◽  
Vol 48 (17) ◽  
pp. 9681-9693
Author(s):  
Zhenzhen Liu ◽  
Mengmeng Sun ◽  
Jilin Liu ◽  
Tao Liu ◽  
Qing Ye ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Genomic Dna ◽  
Cell Aggregation ◽  
Gene Promoters ◽  
Dna Damages ◽  
Associated Factor ◽  
Sulfolobus Islandicus ◽  
Repair Systems

Abstract CRISPR−Cas system provides acquired immunity against invasive genetic elements in prokaryotes. In both bacteria and archaea, transcriptional factors play important roles in regulation of CRISPR adaptation and interference. In the model Crenarchaeon Sulfolobus islandicus, a CRISPR-associated factor Csa3a triggers CRISPR adaptation and activates CRISPR RNA transcription for the immunity. However, regulation of DNA repair systems for repairing the genomic DNA damages caused by the CRISPR self-immunity is less understood. Here, according to the transcriptome and reporter gene data, we found that deletion of the csa3a gene down-regulated the DNA damage response (DDR) genes, including the ups and ced genes. Furthermore, in vitro analyses demonstrated that Csa3a specifically bound the DDR gene promoters. Microscopic analysis showed that deletion of csa3a significantly inhibited DNA damage-induced cell aggregation. Moreover, the flow cytometry study and survival rate analysis revealed that the csa3a deletion strain was more sensitive to the DNA-damaging reagent. Importantly, CRISPR self-targeting and DNA transfer experiments revealed that Csa3a was involved in regulating Ups- and Ced-mediated repair of CRISPR-damaged host genomic DNA. These results explain the interplay between Csa3a functions in activating CRISPR adaptation and DNA repair systems, and expands our understanding of the lost link between CRISPR self-immunity and genome stability.

scholarly journals Activated IGF-1R inhibits hyperglycemia-induced DNA damage and promotes DNA repair by homologous recombination

2005 ◽  
Vol 289 (5) ◽  
pp. F1144-F1152 ◽  
Author(s):  
Shuo Yang ◽  
Janaki Chintapalli ◽  
Lakshmi Sodagum ◽  
Stuart Baskin ◽  
Ashwani Malhotra ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Genomic Dna ◽  
Mesangial Cells ◽  
Reactive Oxygen ◽  
Genotoxic Stress ◽  
Ros Generation ◽  
Oxygen Intermediates ◽  
Strand Breaks

The IGF-1R is a genetic determinant of oxidative stress and longevity. Hyperglycemia induces an exponential increase in the production of a key danger signal, reactive oxygen intermediates, which target genomic DNA. Here, we report for the first time that ligand activation of the IGF-1R prevents hyperglycemia-induced genotoxic stress and enhances DNA repair, maintaining genomic integrity and cell viability. We performed single gel electrophoresis (comet assay) to evaluate DNA damage in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at high ambient glucose concentration. Hyperglycemia inflicted an impressive array of DNA damage in the form of single-strand breaks (SSBs) and double-strand breaks (DSBs). The inclusion of IGF-1 to culture media of MMC and NHMC prevented hyperglycemia-induced DNA damage. To determine whether DNA damage was mediated by reactive oxygen species (ROS), ROS generation was evaluated, in the presence of IGF-1, or the free radical scavenger n-acetyl-cysteine (NAC). IGF-1 and NAC inhibited hyperglycemic-induced ROS production and hyperglycemia-induced DNA damage. We next asked whether IGF-1 promotes the repair of DSB under hyperglycemic conditions, by homologous recombination (HRR) or nonhomologous end joining (NHEJ). Repair of DSB by NHEJ and HRR was operative in MMC maintained under hyperglycemic conditions. IGF-1 increased HRR by nearly twofold, whereas IGF-1 did not affect DNA repair by NHEJ. IGF-1R enhancement of HRR correlated with the translocation of Rad51 to foci of DNA damage. Inhibition of Rad51 expression by short interfering RNA experiments markedly decreased percentage of MMC positive for Rad51 nuclear foci and increased hyperglycemic DNA damage. We conclude that the activated IGF-1R rescues mesangial cells from hyperglycemia-induced danger signals that target genomic DNA by suppressing ROS and enhancing DNA repair by HRR.

Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells

1989 ◽  
Vol 9 (2) ◽  
pp. 851-853
Author(s):  
A J Fornace ◽  
B Zmudzka ◽  
M C Hollander ◽  
S H Wilson
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Repair Gene ◽  
Ovary Cells ◽  
Polymerase Beta ◽  
Dna Damaging Agents ◽  
Dna Repair Gene

Only a few of the genes involved in DNA repair in mammalian cells have been isolated, and induction of a DNA repair gene in response to DNA damage has not yet been established. DNA polymerase beta (beta-polymerase) appears to have a synthetic role in DNA repair after certain types of DNA damage. Here we show that the level of beta-polymerase mRNA is increased in CHO cells after treatment with several DNA-damaging agents.

scholarly journals Nonhomologous-End-Joining Factors Regulate DNA Repair Fidelity during Sleeping Beauty Element Transposition in Mammalian Cells

2003 ◽  
Vol 23 (23) ◽  
pp. 8505-8518 ◽  
Author(s):  
Stephen R. Yant ◽  
Mark A. Kay
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Mammalian Cells ◽  
Donor Site ◽  
Protective Role ◽  
Sleeping Beauty ◽  
Nonhomologous End Joining ◽  
Dna Breaks ◽  
End Joining ◽  
Transposon Insertion

ABSTRACT Herein, we report that the DNA-dependent protein kinase (DNA-PK) regulates the DNA damage introduced during Sleeping Beauty (SB) element excision and reinsertion in mammalian cells. Using both plasmid- and chromosome-based mobility assays, we analyzed the repair of transposase-induced double-stranded DNA breaks in cells deficient in either the DNA-binding subunit of DNA-PK (Ku) or its catalytic subunit (DNA-PKcs). We found that the free 3′ overhangs left after SB element excision were efficiently and accurately processed by the major Ku-dependent nonhomologous-end-joining pathway. Rejoining of broken DNA molecules in the absence of Ku resulted in extensive end degradation at the donor site and greatly increased the frequency of recombination with ectopic templates. Therefore, the major DNA-PK-dependent DNA damage response predominates over more-error-prone repair pathways and thereby facilitates high-fidelity DNA repair during transposon mobilization in mammalian cells. Although transposable elements were not found to be efficiently circularized after transposase-mediated excision, DNA-PK deficiency supported more-frequent transposase-mediated element insertion than was found in wild-type controls. We conclude that, based on its ability to regulate excision site junctional diversity and transposon insertion frequency, DNA-PK serves an important protective role during transpositional recombination in mammals.

scholarly journals ZGRF1 promotes end resection of DNA homologous recombination via forming complex with BRCA1/EXO1

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Shuang Yan ◽  
Man Song ◽  
Jie Ping ◽  
Shu-ting Lai ◽  
Xiao-yu Cao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Mammalian Cells ◽  
Cell Cycle Checkpoint ◽  
Dependent Manner ◽  
Dna Breaks ◽  
M Cell ◽  
End Resection

AbstractTo maintain genomic stability, the mammalian cells has evolved a coordinated response to DNA damage, including activation of DNA repair and cell cycle checkpoint processes. Exonuclease 1 (EXO1)-dependent excision of DNA ends is important for the initiation of homologous recombination (HR) repair of DNA breaks, which is thought to play a key role in activating the ATR-CHK1 pathway to induce G2/M cell cycle arrest. But the mechanism is still not fully understood. Here, we report that ZGRF1 forms complexes with EXO1 as well as other repair proteins and promotes DNA repair through HR. ZGRF1 is recruited to DNA damage sites in a MDC1-RNF8-BRCA1 dependent manner. Furthermore, ZGRF1 is important for the recruitment of RPA2 to DNA damage sites and the following ATR-CHK1 mediated G2/M checkpoint in response to irradiation. ZGRF1 null cells show increased sensitivity to many DNA-damaging agents, especially PARPi and irradiation. Collectively,our findings identify ZGRF1 as a novel regulator of DNA end resection and G2/M checkpoint. ZGRF1 is a potential target of radiation and PARPi cancer therapy.

scholarly journals Role of Trypanosoma cruzi nucleoside diphosphate kinase 1 in DNA-damage responses

2019 ◽  
Author(s):  
Chantal Reigada ◽  
Melisa Sayé ◽  
Fabio Di Girolamo ◽  
Edward A. Valera-Vera ◽  
Claudio A. Pereira ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Dna Repair ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Spontaneous Mutation ◽  
Nucleoside Diphosphate Kinase ◽  
Dna Integrity ◽  
Dna Damage Responses

AbstractNME23/NDPK proteins are well conserved proteins found in all living organism. Besides their catalytic activity of nucleoside diphosphate kinase (NDPK) they are considered multifunctional, which were first characterized as non-metastatic proteins in mammalian cells. Later, increasing evidences placed NME/NDPK as proteins involved in DNA stability such as gene regulation and DNA-repair. TcNDPK1 is the canonical NDPK isoform present in the parasite Trypanosoma cruzi, orthologous to NME23-H1/H2 which has been shown to have in vitro nuclease activity and DNA-binding properties. In the present study we investigate the role of TcNDPK1 in DNA-damage responses using heterologous gene expression systems and over-expression in epimastigote cells. We found that different strains of bacteria, WT and ndk-mutants, expressing the enzyme decreased about 5 fold and 18 fold the spontaneous mutation rate, respectively. In addition, yeasts lacking the endogenous gene YNK1 (YNK1-) and expressing TcNDPK1, were significantly more resistant to different concentrations of hydrogen peroxide and were less sensible to UV radiation than controls. Parasites over-expressing TcNDPK1 were able to withstand different genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. In addition, under oxidative damage, TcNDPK1 over-expressing parasites presented lesser genomic damage and augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair. These results strongly suggest that TcNDPK1 is involved in the maintenance of parasite genomic-DNA integrity, thus, giving rise to a novel function.

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