Application of Pulsed Field Gel Electrophoresis to Determine γ-ray-induced Double-strand Breaks in Yeast Chromosomal Molecules

Friedl, Anna A.; Beisker, Wolfgang; Hahn, Kristen R.; Eckardt‐Schupp, Friederike; Kellerer, Albrecht M.

doi:10.1080/09553009314550231

Cited by 15 publications

(7 citation statements)

References 23 publications

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“…To ensure that MAG1 deletion selectively enhanced sensitivity to MMS, SPY1102 ( mag1 Δ) was exposed to multiple doses of γ-ray, whose most lethal effects are the result of double strand breaks (Friedl et al, 1993). The responses of SPY1101 ( MAG1 ) and SPY1102 ( mag1 Δ) were indistinguishable at every dose tested (Fig.…”

Section: Resultsmentioning

confidence: 99%

Deletion of MAG1 and MRE11 enhances the sensitivity of the Saccharomyces cerevisiae HUG1P-GFP promoter-reporter construct to genotoxicity

Benton

Glasser

Palecek

2008

Biosensors and Bioelectronics

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Eukaryotic yeast-based DNA damage cellular sensors offer many advantages to traditional prokaryotic-based mutagenicity assays. The HUG1P-GFP promoter-reporter construct has proven to be an effective method to selectively screen for multiple types of DNA damage. To enhance the sensitivity and selectivity of the system to different types of DNA damage, two genes involved in distinct DNA damage responses were deleted. Deletion of MAG1, a gene encoding a DNA glycosylase and member of the base excision repair (BER) pathway, increased the biosensor's sensitivity to the alkylating agents methyl methanesulfonate (MMS) (lowering the sensitivity threshold to 0.0001% (v/v)) and ethyl methanesulfonate (EMS). Deletion of MRE11, part of the highly conserved RMX complex that aids in sensing and repairing double strand breaks in budding yeasts, enhanced sensitivity to gamma radiation (γ-ray) (detection threshold of 50 Gy) and camptothecin. The mre11Δ phenotype dominated in mag1Δ mre11Δ strains. Through the deletions, we were able to engineer increased selectivity to alkylating agents, γ-ray, and camptothecin, since increased sensitivity to one type of damage did not alter the quantitative response to other genotoxins. The enhancements to the HUG1P-GFP system did not affect its ability to detect several other DNA damaging agents, including 1,2-dimethyl hydrazine (SDMH), phleomycin, and hydroxyurea (HU), or affect its lack of response to the potentially non-genotoxic carcinogen formaldehyde.

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Section: Resultsmentioning

confidence: 99%

Deletion of MAG1 and MRE11 enhances the sensitivity of the Saccharomyces cerevisiae HUG1P-GFP promoter-reporter construct to genotoxicity

Benton

Glasser

Palecek

2008

Biosensors and Bioelectronics

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“…Mbp were determined by a comparison of their electrophoretic mobility with that of h concatemeric DNA molecules on various gels under different electrophoresis conditions [7]. The lengths of the two longest chromosome species (chromosomes IV and XII) were estimated according to published values [12, 131. Note that the two chromosome XI1 homologs in the strain BKO differ substantially in length; their apparent sizes were estimated as 2.25 Mbp and 3 Mbp.…”

Section: Size Standardsmentioning

confidence: 99%

“…Measurement of the distribution of fluorescence intensity along the lanes of an ethidium bromide-stained gel then yields profiles which can be regarded as distributions of DNA mass as functions of migration distance. Our first approach is based on a technique successfully applied to the DSB quantification in yeast [7,81, where calculations are performed aiming at the prediction of profiles which correspond well to the observed ones. In these calculations, which assume a random distribution of DSB and require an adequate description of the migration behavior of the DNA molecules during electrophoresis, the DSB frequency is a free input parameter, whose value has to be adjusted until the best agreement between calculated and observed profile is obtained.…”

Section: Introductionmentioning

confidence: 99%

An electrophoretic approach to the assessment of the spatial distribution of DNA double‐strand breaks in mammalian cells

Friedl¹,

Kraxenberger²,

Eckardt‐Schupp³

1995

Electrophoresis

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An approach is presented making it possible to investigate whether breaks in fragmented mammalian chromosomal DNA were induced randomly and independently from each other. Genomic DNA isolated from mammalian cells irradiated with gamma-rays or restriction enzyme-treated human DNA was resolved according to size using pulsed field gel electrophoresis, and the resulting DNA mass distributions were measured in ethidium bromide-stained gels. The DNA profiles thus obtained were compared to the predictions on DNA fragment size distribution which follow from a so-called random breakage model to test whether the experimental outcome is compatible with the assumption of a random localization of breaks. Comparisons of fragment distributions may be performed utilizing two equivalent representations that are linked by an adequate transformation. Considering either directly measurable DNA mass profiles in units of migration distances along a gel lane or transformed distributions in units of molecular length, we show for gamma-irradiated samples that the predictions derived from the employed models agree well with the observed data, thus allowing an immediate quantification of double-strand breaks (DSB). Using restriction enzyme-treated DNA as a paradigm, the disagreement of predicted and observed data shows the applicability of our approach to the detection of a non-random distribution of DSB. Therefore, we suppose that our approach may also be useful to reveal a clustering of DSB, which is postulated to occur after damage induction by densely ionizing radiation. Furthermore, investigations on the spatial distribution of chemically or endogenously produced DSB, as well as residual DSB after repair, may be attempted.

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“…These latter lesions inhibit DNA synthesis and must be repaired for replication to properly occur [30,32]. Ionizing radiation induces multiple types of DNA damage, but double strand breaks are considered to be the most lethal [33]. MMS generally produces single-strand lesions which inhibit DNA replication at the S phase checkpoint, while double-strand breaks caused by γ-ray inhibit the G 2 /M checkpoint [34].…”

Section: Introductionmentioning

confidence: 99%

Analyzing the dose-dependence of the Saccharomyces cerevisiae global transcriptional response to methyl methanesulfonate and ionizing radiation

et al. 2006

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Background: One of the most crucial tasks for a cell to ensure its long term survival is preserving the integrity of its genetic heritage via maintenance of DNA structure and sequence. While the DNA damage response in the yeast Saccharomyces cerevisiae, a model eukaryotic organism, has been extensively studied, much remains to be elucidated about how the organism senses and responds to different types and doses of DNA damage. We have measured the global transcriptional response of S. cerevisiae to multiple doses of two representative DNA damaging agents, methyl methanesulfonate (MMS) and gamma radiation.

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Application of Pulsed Field Gel Electrophoresis to Determine γ-ray-induced Double-strand Breaks in Yeast Chromosomal Molecules

Abstract: Application of programmable, autonomously controlled electrode (PACE) technology to the development of an improved pulsed field gel electrophoresis assay for DNA double-strand breaks in mammalian cells (

Cited by 15 publications

References 23 publications

Deletion of MAG1 and MRE11 enhances the sensitivity of the Saccharomyces cerevisiae HUG1P-GFP promoter-reporter construct to genotoxicity

Deletion of MAG1 and MRE11 enhances the sensitivity of the Saccharomyces cerevisiae HUG1P-GFP promoter-reporter construct to genotoxicity

An electrophoretic approach to the assessment of the spatial distribution of DNA double‐strand breaks in mammalian cells

Analyzing the dose-dependence of the Saccharomyces cerevisiae global transcriptional response to methyl methanesulfonate and ionizing radiation

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