Analysis of DNA damage and repair in saccharomyces cerevisiae using the comet assay in the characterization of antigenotoxicity of plant extracts and phytochemicals

Abstract

In this work we used the model organism Saccharomyces cerevisiae to characterise the biological activity and the mechanism of action of phytochemicals. One of the goals is to use mutant strains affected in basic mechanisms of oxidative stress response and DNA repair in order to uncover the molecular targets of phytochemicals. We have assessed DNA damage and repair using the comet assay, evaluated as “comet tail length”, which displayed a dose-response relationship with different DNA-damaging agents1. Subsequently, we used this system to assess the antigenotoxic properties of a leaf extract from Ginkgo biloba (GBE). Typical experiments involved incubation of yeast cells, or spheroplasts, with GBE before and during oxidative shock with hydrogen peroxide. Our results obtained with the comet assay show that DNA damage was significantly decreased upon GBE treatment in a dose-dependent manner. In addition, DNA repair kinetics was significantly improved in cells incubated with GBE. However, in the mutant strain affected in CDC9, encoding a DNA ligase involved in the mechanisms of nucleotide excision repair and base excision repair, oxidative DNA damage repair kinetics was unchanged with GBE, suggesting that the activity of this extract involves one of these mechanisms, or both. Hydrogen peroxide-induced cell cycle arrest in G2 was abolished when cells were incubated with GBE after oxidative shock, suggesting that the improved repair kinetics allows progression of the cell cycle and/or GBE can have a direct effect on its regulation. As expected, GBE treatment improved survival of yeast cells when challenged with oxidative shock with H2O2 and intracellular oxidation was considerably decreased upon pre-treatment with GBE as revealed by flow cytometry. Reference(s) 1. Azevedo F., Marques, F., Fokt, H., Oliveira, R. and Johansson, B. (2011) Measuring oxidative DNA damage and DNA repair using the yeast comet assay. Yeast, 28, 55-61