m0308s  (New England Biolabs)

Journal: Antioxidants

Article Title: Rhus coriaria L. Fruit Extract Prevents UV-A-Induced Genotoxicity and Oxidative Injury in Human Microvascular Endothelial Cells

doi: 10.3390/antiox9040292

Figure Lengend Snippet: Characterization of genotoxic damage induced by UV-A, and mERC extract’s role. Modified Comet assay measured direct DNA damage using ( a ) T4 PDG enzyme recognizing CPDs; indirect i.e., oxidative damage was identified by ( b ) ENDO III for oxidized pyrimidines or ( c ) FPG for oxidized purines. HMEC-1 cells were treated for 1 h with mERC (25 μg/mL, E25) and exposed to 20 J/cm 2 UV-A (T20). Results are expressed as mean of medians ± SEM, n = 3. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. ### p

Article Snippet: The alkaline Comet assay was performed with some variations: the pH of the alkaline buffer was set at 12.1; after the lysis, slides were incubated for 45 min at 37 °C with 50 μL of the diluted enzymes: T4 PDG, ENDO III, FPG (New England BioLabs® Inc., 75-77 Knowl Piece, Wilbury Wai, Hitchin, UK).

Techniques: Modification, Single Cell Gel Electrophoresis

Journal: Nature Communications

Article Title: Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions

doi: 10.1038/s41467-019-09146-5

Figure Lengend Snippet: In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by T4-PDG. Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively

Article Snippet: Formation of T–T dimer were determined from the sensitivity of the UV-exposed template to the enzyme T4-PDG (NEB, USA).

Techniques: In Vitro, Generated, Nucleic Acid Electrophoresis, Irradiation, Labeling