scholarly journals XPD/ERCC2 mutations interfere in cellular responses to oxidative stress

Mutagenesis ◽  
2019 ◽  
Vol 34 (4) ◽  
pp. 341-354 ◽  
Author(s):  
Leticia K Lerner ◽  
Natália C Moreno ◽  
Clarissa R R Rocha ◽  
Veridiana Munford ◽  
Valquíria Santos ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Lines ◽  
Excision Repair ◽  
Dna Lesions ◽  
Repair Capacity ◽  
Strand Breaks ◽  
Nucleotide Excision ◽  
Dna Strand ◽  
Ultraviolet Damage ◽  
Host Cell Reactivation

Abstract Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPD-mutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell reactivation (HCR) assays showed that XP-D/CS and TTD cells have severely impaired repair capacity of oxidised lesions in plasmid DNA, and alkaline comet assays demonstrated the induction of significantly higher amounts of DNA strand breaks after treatment with photoactivated MB in these cells compared to wild-type cells. All XPD-mutated cells presented strong S/G2 arrest and persistent γ-H2AX staining after photoactivated MB treatment. Taken together, these results indicate that XPD participates in the repair of lesions induced by the redox process, and that XPD mutations lead to differences in the response to oxidatively induced damage.

scholarly journals Elevated glucose increases genomic instability by inhibiting nucleotide excision repair

2021 ◽  
Vol 4 (10) ◽  
pp. e202101159
Author(s):  
Alexandra K Ciminera ◽  
Sarah C Shuck ◽  
John Termini
Keyword(s):  
Dna Repair ◽  
Genomic Instability ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Hypoxia Inducible Factor ◽  
Strand Break ◽  
Strand Breaks ◽  
Nucleotide Excision ◽  
Elevated Glucose ◽  
Dna Strand

We investigated potential mechanisms by which elevated glucose may promote genomic instability. Gene expression studies, protein measurements, mass spectroscopic analyses, and functional assays revealed that elevated glucose inhibited the nucleotide excision repair (NER) pathway, promoted DNA strand breaks, and increased levels of the DNA glycation adduct N2-(1-carboxyethyl)-2ʹ-deoxyguanosine (CEdG). Glycation stress in NER-competent cells yielded single-strand breaks accompanied by ATR activation, γH2AX induction, and enhanced non-homologous end-joining and homology-directed repair. In NER-deficient cells, glycation stress activated ATM/ATR/H2AX, consistent with double-strand break formation. Elevated glucose inhibited DNA repair by attenuating hypoxia-inducible factor-1α–mediated transcription of NER genes via enhanced 2-ketoglutarate–dependent prolyl hydroxylase (PHD) activity. PHD inhibition enhanced transcription of NER genes and facilitated CEdG repair. These results are consistent with a role for hyperglycemia in promoting genomic instability as a potential mechanism for increasing cancer risk in metabolic disease. Because of the pleiotropic functions of many NER genes beyond DNA repair, these results may have broader implications for cellular pathophysiology.

Effect of nucleotide excision repair in human cells on intrachromosomal homologous recombination induced by UV and 1-nitrosopyrene

1990 ◽  
Vol 10 (8) ◽  
pp. 3945-3951
Author(s):  
N P Bhattacharyya ◽  
V M Maher ◽  
J J McCormick
Keyword(s):  
Homologous Recombination ◽  
Thymidine Kinase ◽  
Nucleotide Excision Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Dna Lesions ◽  
Repair Capacity ◽  
Nucleotide Excision ◽  
Specific Increase ◽  
Simplex Virus

To study the role of nucleotide excision repair in the induction of intrachromosomal homologous recombination in mammalian cells, we introduced a plasmid containing a substrate for recombination into three human cell lines that differ in their repair capacity and compared the frequency of recombination induced by UV radiation and by 1-nitrosopyrene. One strain had a normal capacity for nucleotide excision repair, the second exhibited an intermediate rate of repair, and the third, derived from a patient with xeroderma pigmentosum, had no ability to repair UV- or 1-nitrosopyrene-induced DNA damage. The endogenous thymidine kinase genes in these cell strains had been inactivated, and the cells contained an integrated copy of a plasmid carrying duplicated copies of the herpes simplex virus type 1 thymidine kinase (Htk) gene, each inactivated by an 8-base-pair XhoI site inserted at a unique site. A functional tk gene can only be generated by a productive recombination event between the two Htk genes. In all three stains, UV and 1-nitrosopyrene induced dose-dependent increases in the frequency of recombinants. However, the doses required to cause a specific increase in recombination in the repair-deficient strains were 10 to 30 times lower than the dose required for the cell strain with a normal capacity for repair. These results strongly suggest that unexcised DNA lesions, rather than excision repair per se, stimulate intrachromosomal homologous recombination. Southern blot analysis of DNA from representative recombinants indicated that in all cases one of the two Htk genes had become wild type (XhoI resistant). The majority (90%) retained the Htk duplication, consistent with nonreciprocal transfer of genetic information (gene conversion).

scholarly journals H. pylori -Induced DNA Strand Breaks Are Introduced by Nucleotide Excision Repair Endonucleases and Promote NF-κB Target Gene Expression

Cell Reports ◽  
2015 ◽  
Vol 13 (1) ◽  
pp. 70-79 ◽  
Author(s):  
Mara L. Hartung ◽  
Dorothea C. Gruber ◽  
Katrin N. Koch ◽  
Livia Grüter ◽  
Hubert Rehrauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nucleotide Excision Repair ◽  
Target Gene ◽  
Excision Repair ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Target Gene Expression ◽  
Nucleotide Excision ◽  
Dna Strand ◽  
H Pylori

scholarly journals The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection

2021 ◽  
Vol 4 (1) ◽  
pp. 14
Author(s):  
Maria Rita Fabbrizi ◽  
Jonathan R. Hughes ◽  
Jason L. Parsons
Keyword(s):  
Dna Damage ◽  
Complex Analysis ◽  
Single Cells ◽  
Strand Break ◽  
Dna Lesions ◽  
Repair Capacity ◽  
Strand Breaks ◽  
Dna Strand Break ◽  
Repair Enzymes ◽  
Dna Strand

The comet assay is a versatile, simple, and sensitive gel electrophoresis–based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. While generally this is used to measure variation in DNA strand break levels and repair capacity within a population of cells, the technique has more recently been adapted and evolved into more complex analysis and detection of specific DNA lesions, such as oxidized purines and pyrimidines, achieved through the utilization of damage-specific DNA repair enzymes following cell lysis. Here, we detail a version of the enzyme-modified neutral comet (EMNC) assay for the specific detection of complex DNA damage (CDD), defined as two or more DNA damage lesions within 1–2 helical turns of the DNA. CDD induction is specifically relevant to ionizing radiation (IR), particularly of increasing linear energy transfer (LET), and is known to contribute to the cell-killing effects of IR due to the difficult nature of its repair. Consequently, the EMNC assay reveals important details regarding the extent and complexity of DNA damage induced by IR, but also has potential for the study of other genotoxic agents that may induce CDD.

scholarly journals Functions of the CSB Protein at Topoisomerase 2 Inhibitors-Induced DNA Lesions

2021 ◽  
Vol 9 ◽  
Author(s):  
Franciele Faccio Busatto ◽  
Sofiane Y. Mersaoui ◽  
Yilun Sun ◽  
Yves Pommier ◽  
Jean-Yves Masson ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Excision Repair ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Nucleotide Excision ◽  
Different Types ◽  
Topoisomerase 2 ◽  
Nucleotide Excision Repair Pathway

Topoisomerase 2 (TOP2) inhibitors are drugs widely used in the treatment of different types of cancer. Processing of their induced-lesions create double-strand breaks (DSBs) in the DNA, which is the main toxic mechanism of topoisomerase inhibitors to kill cancer cells. It was established that the Nucleotide Excision Repair pathway respond to TOP2-induced lesions, mainly through the Cockayne Syndrome B (CSB) protein. In this paper, we further define the mechanism and type of lesions induced by TOP2 inhibitors when CSB is abrogated. In the absence of TOP2, but not during pharmacological inhibition, an increase in R-Loops was detected. We also observed that CSB knockdown provokes the accumulation of DSBs induced by TOP2 inhibitors. Consistent with a functional interplay, interaction between CSB and TOP2 occurred after TOP2 inhibition. This was corroborated with in vitro DNA cleavage assays where CSB stimulated the activity of TOP2. Altogether, our results show that TOP2 is stimulated by the CSB protein and prevents the accumulation of R-loops/DSBs linked to genomic instability.

scholarly journals Effect of nucleotide excision repair in human cells on intrachromosomal homologous recombination induced by UV and 1-nitrosopyrene.

1990 ◽  
Vol 10 (8) ◽  
pp. 3945-3951 ◽  
Author(s):  
N P Bhattacharyya ◽  
V M Maher ◽  
J J McCormick
Keyword(s):  
Homologous Recombination ◽  
Thymidine Kinase ◽  
Nucleotide Excision Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Dna Lesions ◽  
Repair Capacity ◽  
Nucleotide Excision ◽  
Specific Increase ◽  
Simplex Virus

To study the role of nucleotide excision repair in the induction of intrachromosomal homologous recombination in mammalian cells, we introduced a plasmid containing a substrate for recombination into three human cell lines that differ in their repair capacity and compared the frequency of recombination induced by UV radiation and by 1-nitrosopyrene. One strain had a normal capacity for nucleotide excision repair, the second exhibited an intermediate rate of repair, and the third, derived from a patient with xeroderma pigmentosum, had no ability to repair UV- or 1-nitrosopyrene-induced DNA damage. The endogenous thymidine kinase genes in these cell strains had been inactivated, and the cells contained an integrated copy of a plasmid carrying duplicated copies of the herpes simplex virus type 1 thymidine kinase (Htk) gene, each inactivated by an 8-base-pair XhoI site inserted at a unique site. A functional tk gene can only be generated by a productive recombination event between the two Htk genes. In all three stains, UV and 1-nitrosopyrene induced dose-dependent increases in the frequency of recombinants. However, the doses required to cause a specific increase in recombination in the repair-deficient strains were 10 to 30 times lower than the dose required for the cell strain with a normal capacity for repair. These results strongly suggest that unexcised DNA lesions, rather than excision repair per se, stimulate intrachromosomal homologous recombination. Southern blot analysis of DNA from representative recombinants indicated that in all cases one of the two Htk genes had become wild type (XhoI resistant). The majority (90%) retained the Htk duplication, consistent with nonreciprocal transfer of genetic information (gene conversion).

scholarly journals Endonuclease IV Is the Main Base Excision Repair Enzyme Involved in DNA Damage Induced by UVA Radiation and Stannous Chloride

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Ellen S. Motta ◽  
Paulo Thiago Souza-Santos ◽  
Tuany R. Cassiano ◽  
Flávio J. S. Dantas ◽  
Adriano Caldeira-de-Araujo ◽  
...  
Keyword(s):  
Stannous Chloride ◽  
Excision Repair ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Lesion ◽  
Base Excision ◽  
Dna Strand ◽  
Uva Radiation

Stannous chloride (SnCl2) and UVA induce DNA lesions through ROS. The aim of this work was to study the toxicity induced by UVA preillumination, followed bySnCl2treatment.E. coliBER mutants were used to identify genes which could play a role in DNA lesion repair generated by these agents. The survival assays showed (i) Thenfomutant was the most sensitive toSnCl2; (ii) lethal synergistic effect was observed after UVA pre-illumination, plusSnCl2incubation, thenfomutant being the most sensitive; (iii) wild type andnfomutants, transformed with pBW21 plasmid (nfo+) had their survival increased following treatments. The alkaline agarose gel electrophoresis assays pointed that (i) UVA induced DNA breaks andfpgmutant was the most sensitive; (ii)SnCl2-induced DNA strand breaks were higher than those from UVA andnfomutant had the slowest repair kinetics; (iii)UVA+SnCl2promoted an increase in DNA breaks thanSnCl2and, again,nfomutant displayed the slowest repair kinetics. In summary, Nfo protectsE. colicells against damage induced bySnCl2andUVA+SnCl2.

Sign in / Sign up

Export Citation Format

Share Document