Detection of DNA Damage in Prokaryotes by Terminal Deoxyribonucleotide Transferase-Mediated dUTP Nick End Labeling

ABSTRACT Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3′-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3′-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeonHaloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3′-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E. coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.

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Preferential repair of UV damage in highly transcribed DNA diminishes UV-induced intrachromosomal recombination in mammalian cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.391-399.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 391-399

Author(s):

W P Deng ◽

J A Nickoloff

Keyword(s):

Dna Damage ◽

Mammalian Cells ◽

Uv Light ◽

Uv Damage ◽

Dna Damage And Repair ◽

Intrachromosomal Recombination ◽

Dna Damaging Agents ◽

Mouse Mammary Tumor ◽

Tumor Virus ◽

Virus Promoter

The relationships among transcription, recombination, DNA damage, and repair in mammalian cells were investigated. We monitored the effects of transcription on UV-induced intrachromosomal recombination between neomycin repeats including a promoterless allele and an inducible heteroallele regulated by the mouse mammary tumor virus promoter. Although transcription and UV light separately stimulated recombination, increasing transcription levels reduced UV-induced recombination. Preferential repair of UV damage in transcribed strands was shown in highly transcribed DNA, suggesting that recombination is stimulated by unrepaired UV damage and that increased DNA repair in highly transcribed alleles removes recombinogenic lesions. This study indicates that the genetic consequences of DNA damage depend on transcriptional states and provides a basis for understanding tissue- and gene-specific responses to DNA-damaging agents.

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Preferential repair of UV damage in highly transcribed DNA diminishes UV-induced intrachromosomal recombination in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.391 ◽

1994 ◽

Vol 14 (1) ◽

pp. 391-399 ◽

Cited By ~ 18

Author(s):

W P Deng ◽

J A Nickoloff

Keyword(s):

Dna Damage ◽

Mammalian Cells ◽

Uv Light ◽

Uv Damage ◽

Dna Damage And Repair ◽

Intrachromosomal Recombination ◽

Dna Damaging Agents ◽

Mouse Mammary Tumor ◽

Tumor Virus ◽

Virus Promoter

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Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3314-3322.1989 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3314-3322

Author(s):

G M Cole ◽

R K Mortimer

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Dna Repair ◽

Constitutive Expression ◽

Inducible Promoter ◽

Positive Control ◽

Repair Gene ◽

Dna Damaging Agents ◽

Dna Repair Gene ◽

Mating Type Switching

The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNA-damaging agents. Induction of RAD54 by DNA-damaging agents is under positive control. Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site. This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression. Deletions which eliminate induction of RAD54 transcription by DNA damage but do not affect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents. The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination. We find no phenotype for a lack of induction of RAD54 message via the damage-inducible DRS, which raises significant questions about the physiology of DNA damage induction in S. cerevisiae.

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Variability in DNA repair and individual susceptibility to genotoxins

Clinical Chemistry ◽

10.1093/clinchem/41.12.1848 ◽

1995 ◽

Vol 41 (12) ◽

pp. 1848-1853 ◽

Cited By ~ 16

Author(s):

S A Kyrtopoulos

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Protective Role ◽

Interindividual Variation ◽

Human Populations ◽

Cytostatic Drugs ◽

Dna Damage And Repair ◽

Cellular Protection ◽

Methylated Dna ◽

Methylating Agents

Abstract DNA repair is an important mechanism of cellular protection from the effects of genotoxic chemicals. Although extensive evidence from studies in experimental systems indicates that variation in DNA repair can significantly influence susceptibility to genotoxins, corresponding studies in human populations are so far limited, mainly because of methodological difficulties. One system, using observations of the accumulation and repair of DNA damage in cancer patients treated with alkylating cytostatic drugs, has provided useful information for assessing the effects of interindividual variation in DNA repair activity on the induction of genotoxic effects in humans. The most detailed studies of this kind have been carried out on patients with cancer (i.e., Hodgkin disease, malignant melanoma) treated with the methylating cytostatic drugs procarbazine or dacarbazine; these studies have provided detailed information on dose-response relationships. They have also demonstrated the protective role of the repair enzyme O6-alkylguanine-DNA alkyltransferase against the accumulation of the premutagenic methylated DNA lesion O6-methylguanine in patients' DNA. Given the strong evidence that exposure of the general population to environmental methylating agents may be extensive, as indicated by the frequent discovery of methylated DNA adducts in human DNA, data on DNA damage and repair in alkylating drug-treated patients and their modulation by host factors may prove useful in efforts to assess the possible carcinogenic risks posed by exposure to environmental methylating agents.

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Mutational signatures are jointly shaped by DNA damage and repair

Nature Communications ◽

10.1038/s41467-020-15912-7 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 19

Author(s):

Nadezda V. Volkova ◽

Bettina Meier ◽

Víctor González-Huici ◽

Simone Bertolini ◽

Santiago Gonzalez ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Excision Repair ◽

Point Mutations ◽

Mutational Signatures ◽

Dna Damage And Repair ◽

C Elegans ◽

Dna Repair Deficiency ◽

Dna Alterations ◽

Repair Pathways

AbstractCells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.

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DNA Repair Biosensor-Identified DNA Damage Activities of Endophyte Extracts from Garcinia cowa

Biomolecules ◽

10.3390/biom10121680 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1680

Author(s):

Tassanee Lerksuthirat ◽

Rakkreat Wikiniyadhanee ◽

Sermsiri Chitphuk ◽

Wasana Stitchantrakul ◽

Somponnat Sampattavanich ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Fluorescent Proteins ◽

Strand Break ◽

Control Group ◽

Focus Formation ◽

Dna Damage And Repair ◽

Biological Compounds ◽

Repair Pathways ◽

Garcinia Cowa

Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.

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The Dark Side of UV-Induced DNA Lesion Repair

Genes ◽

10.3390/genes11121450 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1450

Author(s):

Wojciech Strzałka ◽

Piotr Zgłobicki ◽

Ewa Kowalska ◽

Aneta Bażant ◽

Dariusz Dziga ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Uv Light ◽

Genetic Material ◽

Dna Lesions ◽

Dark Side ◽

Repair System ◽

Strand Breaks ◽

Dna Lesion ◽

Pyrimidine Dimers

In their life cycle, plants are exposed to various unfavorable environmental factors including ultraviolet (UV) radiation emitted by the Sun. UV-A and UV-B, which are partially absorbed by the ozone layer, reach the surface of the Earth causing harmful effects among the others on plant genetic material. The energy of UV light is sufficient to induce mutations in DNA. Some examples of DNA damage induced by UV are pyrimidine dimers, oxidized nucleotides as well as single and double-strand breaks. When exposed to light, plants can repair major UV-induced DNA lesions, i.e., pyrimidine dimers using photoreactivation. However, this highly efficient light-dependent DNA repair system is ineffective in dim light or at night. Moreover, it is helpless when it comes to the repair of DNA lesions other than pyrimidine dimers. In this review, we have focused on how plants cope with deleterious DNA damage that cannot be repaired by photoreactivation. The current understanding of light-independent mechanisms, classified as dark DNA repair, indispensable for the maintenance of plant genetic material integrity has been presented.

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Yeast Rpn4 Links the Proteasome and DNA Repair via RAD52 Regulation

International Journal of Molecular Sciences ◽

10.3390/ijms21218097 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8097

Author(s):

Daria S. Spasskaya ◽

Nonna I. Nadolinskaia ◽

Vera V. Tutyaeva ◽

Yuriy P. Lysov ◽

Vadim L. Karpov ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Proteasome Inhibition ◽

26S Proteasome ◽

Dependent Manner ◽

Dna Repair Genes ◽

Damage Resistance ◽

Dna Damaging Agents ◽

Mutant Strains ◽

Repair Genes

Environmental and intracellular factors often damage DNA, but multiple DNA repair pathways maintain genome integrity. In yeast, the 26S proteasome and its transcriptional regulator and substrate Rpn4 are involved in DNA damage resistance. Paradoxically, while proteasome dysfunction may induce hyper-resistance to DNA-damaging agents, Rpn4 malfunction sensitizes yeasts to these agents. Previously, we proposed that proteasome inhibition causes Rpn4 stabilization followed by the upregulation of Rpn4-dependent DNA repair genes and pathways. Here, we aimed to elucidate the key Rpn4 targets responsible for DNA damage hyper-resistance in proteasome mutants. We impaired the Rpn4-mediated regulation of candidate genes using the CRISPR/Cas9 system and tested the sensitivity of mutant strains to 4-NQO, MMS and zeocin. We found that the separate or simultaneous deregulation of 19S or 20S proteasome subcomplexes induced MAG1, DDI1, RAD23 and RAD52 in an Rpn4-dependent manner. Deregulation of RAD23, DDI1 and RAD52 sensitized yeast to DNA damage. Genetic, epigenetic or dihydrocoumarin-mediated RAD52 repression restored the sensitivity of the proteasome mutants to DNA damage. Our results suggest that the Rpn4-mediated overexpression of DNA repair genes, especially RAD52, defines the DNA damage hyper-resistant phenotype of proteasome mutants. The developed yeast model is useful for characterizing drugs that reverse the DNA damage hyper-resistance phenotypes of cancers.

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Databases and Bioinformatics Tools for the Study of DNA Repair

Molecular Biology International ◽

10.4061/2011/475718 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Kaja Milanowska ◽

Kristian Rother ◽

Janusz M. Bujnicki

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Excision Repair ◽

Uv Light ◽

Dna Lesions ◽

Environmental Chemicals ◽

Nonhomologous End Joining ◽

End Joining ◽

Repair Mechanisms ◽

Base Excision

DNA is continuously exposed to many different damaging agents such as environmental chemicals, UV light, ionizing radiation, and reactive cellular metabolites. DNA lesions can result in different phenotypical consequences ranging from a number of diseases, including cancer, to cellular malfunction, cell death, or aging. To counteract the deleterious effects of DNA damage, cells have developed various repair systems, including biochemical pathways responsible for the removal of single-strand lesions such as base excision repair (BER) and nucleotide excision repair (NER) or specialized polymerases temporarily taking over lesion-arrested DNA polymerases during the S phase in translesion synthesis (TLS). There are also other mechanisms of DNA repair such as homologous recombination repair (HRR), nonhomologous end-joining repair (NHEJ), or DNA damage response system (DDR). This paper reviews bioinformatics resources specialized in disseminating information about DNA repair pathways, proteins involved in repair mechanisms, damaging agents, and DNA lesions.

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