Measuring oxidative DNA damage and DNA repair using the yeast comet assay

Abstract

Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first-order kinetics with a tail length half-life of 11 min at 37• C. This is, to our knowledge, the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage, as shown by a three-fold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair.

Dose-Dependent Protective and Inductive Effects of Xanthohumol on Oxidative DNA Damage in Saccharomyces cerevisiae

“…The genotoxic eff ect did not promote longer comet tails at concentrations above 10 mg/L (Fig. 7), possibly due to the limited capacity of the genomic DNA to unwind and migrate during electrophoresis in the comet assay (30).…”

“…The genome-protective eff ect of xanthohumol against oxidative DNA damage induced by H 2 O 2 was evaluated using the yeast comet assay. The yeast comet assay is a well-established, simple, versatile, sensitive and inexpensive technique used to assess DNA oxidative damage quantitatively and qualitatively in eukaryotic cell populations (30)(31)(32)(33). Yeast spheroplasts were incubated with diff erent xanthohumol concentrations and 10 mM H 2 O 2 in S buff er (containing 1 M sorbitol) in order to maintain osmotic protection.…”

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

“…We followed the procedures described by Azevedo et al [13] with some modifications. A good number of spheroplast with degraded cell wall was obtained by applying 3 mg/ml lyticase at 37°C in water bath for 30 min.…”

Brewer’s Spent Grains Protects against Oxidative DNA Damage in Saccharomyces cerevisiae

“…In a situation of DNA oxidative damage, cell cycle is arrested for the activation of repair mechanisms and if damage cannot be repaired, mechanisms of programmed cell death are activated. The principal DNA repair pathways are base excision repair (BER) by removing oxidation damaged single bases and nucleotide excision repair (NER), which is involved in repairing bulky DNA lesions caused by ultraviolet light (Azevedo, Marques, Fokt, Oliveira, & Johansson, 2011).…”

“…The comet assay has been used for a variety of applications with several organisms (Rank, Syberg, & Jensen, 2009) and was also optimized for application in yeast cells (Saccharomyces cerevisiae) . Azevedo et al (2011) reported an optimized comet assay protocol for yeast cells to evaluate DNA damage and oxidative damage caused by H 2 O 2 with high sensitivity and reproducibility.…”