Measuring oxidative DNA damage and DNA repair using the yeast comet assay.
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.
Azevedo
Flávio
F
Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Marques
Filipe
F
Fokt
Hanna
H
Oliveira
Rui
R
Johansson
Björn
B
D000975:Antioxidants / Q000378:metabolism; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage; D004260:DNA Repair; D004271:DNA, Fungal / Q000378:metabolism*; D006207:Half-Life; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D012441:Saccharomyces cerevisiae / Q000235:genetics
D000975:Antioxidants; D004271:DNA, Fungal; D006861:Hydrogen Peroxide