DNA damage repair ability in cell cycle arrested saccharomyces cerevisiae cells

Abstract

Cells are exposed permanently to different types of environmental stress and to stress caused by oxidative metabolism in mitochondria that target macromolecules like DNA. Although cells dispose a range of stress response mechanisms, balance between DNA damage-causing stress and DNA repair capacity is crucial. Cells have evolved molecular repair mechanisms in case of DNA damage so that genome stability is maintained. Once DNA damage occurs, cell cycle is arrested, allowing the focus of all resources needed for the repair process. Hydroxyurea (HU), a compound that blocks the synthesis of deoxynucleotides, inhibiting DNA synthesis, is able to induce cells to arrest in S phase of the cell cycle. In the present study we aim to investigate the effect of cell cycle arrest on the DNA repair capacity of Saccharomyces cerevisiae cells treated with hydrogen peroxide. We used the comet assay, previously optimized for yeast in our laboratory, in cells grown in medium containing HU and then submitted to oxidative shock. In this test, cells are embedded on agarose, lysed and a short electrophoresis is applied in order to move de genomic DNA out of the nucleoid. The length of the "DNA tail" correlates with DNA damage. Determination of DNA damage and repair was performed through the measurement of the DNA tail length immediately after the oxidative shock and after incubation at 30ºC for different time points, respectively. Also, we have tested HU on cells pre-treated with Ginkgo biloba L. leaves extract (GBE), suggested by previous studies by our group to have an antigenotoxic effect on cells challenged with oxidative shock. Preliminary results obtained show that cells grown in medium containing HU are less susceptible to oxidative stress provoked by different concentrations of H2O2, compared to the control situation. In addition, pre-incubation with GBE improved DNA damage repair. Thus, these results suggest that cells with arrested cell cycle are more efficient in DNA damage repair and less susceptible to oxidative stress. Furthermore, we show evidence suggesting that GBE improves this repair process in cell cycle-arrested cells.