Analysis of alcohol-induced DNA damage in Escherichia coli by visualizing single genomic DNA molecules

Abstract

Consumption of alcohol injures DNA, and such damage is considered to be a primary cause for the development of cancer and many other diseases essentially due to reactive oxygen species generated from alcohol. To sensitively detect alcohol-induced DNA lesions in a biological system, we introduced a novel analytical platform for visualization of single genomic DNA molecules using E. coli. By fluorescently labelling the DNA lesions, our approach demonstrated, with the highest sensitivity, that we could count the number of DNA lesions induced by alcohol metabolism in a single bacterial cell. Moreover, our results showed a linear relationship between ethanol concentration and the number of DNA lesions: 0.88 lesions per 1% ethanol. Using this approach, we quantitatively analysed the DNA damage induced by exposure to alcoholic beverages such as beer (5% ethanol), rice wine (13%), soju (20%), and whisky (40%).

TAMRA-polypyrrole for A/T sequence visualization on DNA molecules

“…In this case, microscopic visualization of the A/T sequence frequency, as shown in Figure 4 , can be an alternative tool to identify the sequence without performing sequencing. Moreover, this approach is particularly useful for analyzing chemically modified or damaged DNA backbones, because conventional sequencing methods can be ineffective on modified or damaged DNA ( 27 , 41 , 43 ). Therefore, microscopic DNA identification offers great promise for further genomic and biochemical studies on single DNA molecules.…”

“…Escherichia coli MG1655 cells were grown at 37°C, 220 rpm, until they reached an OD 600 of 0.5 ( 27 ). After mixing with low melting temperature agarose for 0.75%, 16 U of proteinase K was added to a tube and incubated for 2 h at 42°C.…”

Bioorthogonal Metabolic DNA Labelling using Vinyl Thioether‐Modified Thymidine and o‐Quinolinone Quinone Methide

“…After metabolic incorporation of VTdT,w ei solated genomic DNA in agarose gel according to standard protocol. [15] Then the isolated genomic DNA were incubated in solution containing oQQF for in vitro ligation. Under the same condition, genomic DNA from cells without VTdTi ncubation was also isolated for control experiment.…”

Targets for repair: detecting and quantifying DNA damage with fluorescence-based methodologies

“…Here, we reviewed emerging fluorescence-based techniques for the detection and quantification of DNA damage. While these techniques are not as well-known as mass spectrometry, HPLC, or radiolabeling for adduct detection, they offer new tools for damage characterization that are compatible with other characterization methods, such as live cell imaging and microirradiation [19,31], genomic sequencing [27][28][29], and are capable of being multiplexed with the analysis of other DNA modifications, such as epigenetic marks [30]. While the sensitivity and detection efficiency of these fluorescence-based techniques still require improvement compared to well accepted adduct detection methods (Table 1), overall these offer new opportunities to characterize genotoxicity and examine DNA repair.…”

“…DNA damage is recognized and excised by these enzymes and damage sites are detected by the incorporation of fluorescently-labeled nucleotides by the DNA polymerase. Modified versions of this assay, with different enzyme cocktails, have been utilized to detect oxidative DNA damage in viral genomes [27] and alcohol-induced DNA damage in the E. coli genome [29].…”