Visual quantification of DNA double-strand breaks in bacteria

Singh, Narendra P.; Stephens, R. E.; Singh, Himani; Lai, Henry

doi:10.1016/s0027-5107(99)00124-4

Cited by 38 publications

(32 citation statements)

References 27 publications

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“…We performed neutral single-cell microgel electrophoresis to determine the levels of DSBs in the genomes of wild-type, dam, dam mutS, and mutS mutant cells. Using the single-cell electrophoresis method developed by Singh et al (57,58), we were able to detect a single double-strand break in the genome of a cell. We assume that one linear tail corresponds to a single double-strand break (4), as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Analysis of Global Gene Expression and Double-Strand-Break Formation in DNA Adenine Methyltransferase- and Mismatch Repair-DeficientEscherichia coli

et al. 2005

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DNA adenine methylation by DNA adenine methyltransferase (Dam) in Escherichia coli plays an important role in processes such as DNA replication initiation, gene expression regulation, and mismatch repair. In addition, E. coli strains deficient in Dam are hypersensitive to DNA-damaging agents. We used genome microarrays to compare the transcriptional profiles of E. coli strains deficient in Dam and mismatch repair (dam, dam mutS, and mutS mutants). Our results show that >200 genes are expressed at a higher level in the dam strain, while an additional mutation in mutS suppresses the induction of many of the same genes. We also show by microarray and semiquantitative real-time reverse transcription-PCR that both dam and dam mutS strains show derepression of LexA-regulated SOS genes as well as the up-regulation of other non-SOS genes involved in DNA repair. To correlate the level of SOS induction and the up-regulation of genes involved in recombinational repair with the level of DNA damage, we used neutral single-cell electrophoresis to determine the number of double-strand breaks per cell in each of the strains. We find that dam mutant E. coli strains have a significantly higher level of double-strand breaks than the other strains. We also observe a broad range in the number of double-strand breaks in dam mutant cells, with a minority of cells showing as many as 10 or more double-strand breaks. We propose that the up-regulation of recombinational repair in dam mutants allows for the efficient repair of double-strand breaks whose formation is dependent on functional mismatch repair.The DNA adenine methyltransferase (Dam) protein methylates the N6 position of the adenine residue at d(GATC) sites of the Escherichia coli genome. Dam methylation is a postreplicative process (28), and consequently the newly synthesized daughter strand is unmethylated for a short time after passage of the replication fork. This transient hemimethylated state following DNA replication plays a crucial role in processes such as the regulation of gene expression (11,26,45), DNA mismatch repair (3,45,55), and the timing of chromosome replication initiation (2,24,42,52). By altering the recognition sequences of transcriptional regulators and RNA polymerases, Dam methylation may affect the ability of proteins to bind the upstream regions of genes and in such a way may serve to regulate gene expression. Because d(GATC) sites are not randomly distributed in the E. coli genome (19, 44), Dam deficiency may therefore have a direct effect on gene expression patterns.In methyl-directed mismatch repair, hemimethylated d(GATC) sites serve as the strand discrimination signal so that mismatch repair can differentiate between parent (methylated) and daughter (unmethylated) strands (38). The mismatch repair system relies on three unique proteins: MutS, MutL, and MutH. If there is a misincorporation error following the replication fork, MutS recognizes the mismatch, and a protein-DNA complex is formed with MutS, MutL, and the latent endonuclease MutH. Activat...

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Section: Resultsmentioning

confidence: 99%

“…Cultures were grown as described above. Microgel electrophoresis was performed as described in detail by Singh et al (56)(57)(58). Briefly, small aliquots (0.25 l) of cells were mixed with 50 l of 0.5% agarose (biotechnology grade 3:1; Amresco).…”

Section: Methodsmentioning

confidence: 99%

Analysis of Global Gene Expression and Double-Strand-Break Formation in DNA Adenine Methyltransferase- and Mismatch Repair-DeficientEscherichia coli

et al. 2005

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“…To determine whether the hypersensitivity observed in wildtype cells when compared with the triple mutant lacking the oxidative DNA glycosylases was, in fact, caused by double-strand breaks resulting from attempted repair of clustered lesions, we used neutral microgel electrophoresis to visualize the doublestrand breaks (33). Here, the irradiated cells were gently lysed on a microscope slide, subjected to electrophoresis to ''electrostretch'' the DNA, and the number of double-strand breaks visualized by an intense f luorescent dye, YOYO-1, were counted.…”

Section: Resultsmentioning

confidence: 99%

“…Cultures of exponentially growing cells were starved and irradiated with 0, 45, or 90 Gy as described and then incubated in PBS for either 0, 4, or 8 min at 37°C. After incubation, cultures immediately were cooled to 4°C, and 5 l of a dilution from each culture was embedded in 50 l of lowmelting-temperature agarose and transferred to an MGE microscope slide (Erie Scientific, Portsmouth, NH) as described (32,33). The slides were electrophoresed in neutral conditions for 1 h at 12 volts (Ϸ100 mA) in a modified electrophoretic unit (32) with buffer recirculated at Ϸ100 ml͞min.…”

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Abortive base-excision repair of radiation-induced clustered DNA lesions in Escherichia coli

Blaisdell

Wallace

2001

Proc. Natl. Acad. Sci. U.S.A.

204

112

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It has been postulated that ionizing radiation produces a unique form of cellular DNA damage called ''clustered damages'' or ''multiply damaged sites''. Here, we show that clustered DNA damages are indeed formed in Escherichia coli by ionizing radiation and are converted to lethal double-strand breaks during attempted baseexcision repair. In wild-type cells possessing the oxidative DNA glycosylases that cleave DNA at repairable single damages, doublestrand breaks are formed at radiation-induced clusters during postirradiation incubation and also in a dose-dependent fashion. E. coli mutants lacking these enzymes do not form double-strand breaks postirradiation and are substantially more radioresistant than wild-type cells. Furthermore, overproduction of one of the oxidative DNA glycosylases in mutant cells confers a radiosensitive phenotype and an increase in the number of double-strand breaks. Thus, the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.A pproximately 70% of radiation-induced DNA damages are formed by reactive-free radicals produced by the radiolysis of water in the vicinity of DNA (1). These radiation-induced DNA damages are repaired by base-excision repair (2) and overlap substantially with those formed during normal oxidative metabolism (3), which are produced at significant rates in unirradiated cells (4). This overlap of damages has led to a controversy with respect to determining radiation risk. Proponents of a ''threshold effect'' claim that because endogenous oxidative damages are effectively repaired and adaptive responses have been demonstrated for certain radiation endpoints, then a threshold must exist for the carcinogenic and lethal consequences of ionizing radiation (5, 6). This issue is an important one because much of the projected radiation exposures associated with human activity over the next hundred years will come from low doses associated with medical tests, waste cleanup, and materials associated with nuclear weapons and nuclear power. Whether a threshold exists for the consequences of radiation damage will significantly influence risk estimates for low-dose exposures. Currently, risk estimates for individuals or groups exposed to low radiation doses are determined from epidemiological data obtained from populations exposed to high doses, and a ''linear-no-threshold'' estimation is used for assessment. Proponents of the linear-no-threshold model hold that the biologically important radiation damages in DNA, such as double-strand breaks, are substantially different from single, repairable oxidative lesions. It also has been predicted that ionizing radiation produces a unique form of DNA damage called ''clustered damages '' (1, 7, 8). If, indeed, clustered damages can be demonstrated in living cells and if, unlike the single lesions from which they are formed, they are poorly repaired, these facts would provide additional support for the linear-nothreshold model.Modeling of radiation track structures (9, 10) ...

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“…These technical factors make this procedure impractical for routine assessment of DNA fragmentation in the standard microbiology laboratory. For the comet assay, only one paper has described its use for E. coli (34). The procedure was also lengthy, requiring lysozyme digestion of the cell wall prior to incubation in the lysis solution and in conjunction with prolonged proteinase K digestion.…”

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DNA Fragmentation in Microorganisms Assessed In Situ

Fernández

Cartelle

Muriel

et al. 2008

Appl Environ Microbiol

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Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions.

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Visual quantification of DNA double-strand breaks in bacteria

Cited by 38 publications

References 27 publications

Analysis of Global Gene Expression and Double-Strand-Break Formation in DNA Adenine Methyltransferase- and Mismatch Repair-DeficientEscherichia coli

Analysis of Global Gene Expression and Double-Strand-Break Formation in DNA Adenine Methyltransferase- and Mismatch Repair-DeficientEscherichia coli

Abortive base-excision repair of radiation-induced clustered DNA lesions in Escherichia coli

DNA Fragmentation in Microorganisms Assessed In Situ

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