The Ashbya gossypii comet assay: measuring oxidative and non-oxidative damage in the DNA

Abstract

[Introduction] The Comet Assay (Single Cell Gel Electrophoresis) is one of the most used techniques for measuring DNA damage1 due to its simplicity, sensitivity and versatility2. The theoretical scientific basis of this methodology consists on the increased electrophoretic mobility of damaged DNA. It has a broad range of applications such as: compounds’ genotoxicity/protective testing, DNA repair studies and ecological monitoring. With a huge amount of optimized protocols for superior eukaryotes, the Comet Assay has been poorly utilized among microorganisms. However, Azevedo et al. (2011)3 developed an optimized protocol for Saccharomyces cerevisiae that reinforce the utility and potential of this method for microbial systems. Therefore, in this work we created a reproducible and optimized Comet Assay protocol for use in Ashbya gossypii by adaptation of the Yeast Comet Assay3 (Fig. 1). This protocol allows the measuring of oxidative and non-oxidative DNA damage. Moreover, as riboflavin overproduction in A. gossypii has been associated with stress (mainly oxidative), we went to check if this trait was also associated with increased DNA damage. Therefore, we used this recently created protocol to investigate the DNA damage accumulation in riboflavin overproducing and non-overproducing A. gossypii wild strains.