Studies of DNA repair after an oxidative challenge and its induction by natural compounds : comet assay in Saccharomyces cerevisiae

A reliable and sensitive technique for detection of DNA damage is the Single Cell Gel Assay (also known as comet assay). In this procedure, single cells are exposed to an electric field and damaged DNA moves out of the nucleus, which can be visualized by ethidium bromide staining. Cells display a comet appearance under fluorescence microscopy, the size of the DNA “tail” of the comet being in direct correlation with the extension of DNA damage. In this work we have optimized the comet assay using Saccharomyces cerevisiae cells in order to assess DNA damage caused by oxidative stress. We show that genomic DNA damage increases as the yeast population enters the post-diauxic phase. In addition, the tail comet length decreases by incubation of yeast cells with natural compounds, known to protect cells against oxidative stress (quercetin, ursolic acid), prior to oxidative challenge. Further experiments are being conducted to investigate the DNA repair kinetics by measuring the decrease in the length of the comet tail in a recovery period after an oxidative challenge. We are using mutants affected in RAD1 (an endonuclease of the nucleotide excision repair pathway) and in APN1 (a nuclease of the base excision repair pathway) to determine the repair pathway involved in the oxidative damage. In conclusion, we demonstrate that yeast is a reliable experimental model to screen natural compounds with preventive activity against DNA damage