The importance of vesicular trafficking pathways in genome stability of diploid Saccharomyces cerevisiae cells

The ability to survive stress conditions is important for every living cell. Some stresses can affect not only current cell well-being, but may have far-reaching consequences. Uncurbed oxidative stress can cause DNA damage and the decrease in cell survival and/or increase in mutation rate. Some substances generating oxidative damage in the cell act mainly on DNA. Radiomimetic zeocin is a chemoenzyme that causes oxidative damage in DNA, inducing predominantly single or double strand breaks. Such lesions can subsequently lead to chromosomal rearrangements in genomic DNA, especially in diploid cells in which each sequence has its duplicate in the homologous chromosome. In a global screen for mutants oversensitive to zeocin, we selected 136 genes whose deletion causes the decrease in survival of diploid Saccharomyces cerevisiae cells exposed to this compound. The screen revealed numerous genes connected with stress response, including response to DNA damage stimulus; DNA repair genes, especially connected with homologous recombination and telomere maintenance; genes involved in cell cycle progression, chiefly in control of cell divisions checkpoints, both meiotic and mitotic; and genes involved in remodeling of chromatin. Notably, our screen also demonstrated the involvement of vesicular trafficking system in cell protection against DNA damage. Presented data imply vesicular system in various pathways of cell protection from zeocin-dependent damage, including the role in detoxification and probably more direct role in genome maintenance processes. We show, that cells with vesicular trafficking dysfunction are unable to repair zeocin induced damage, accumulate Rad52 foci and frequently possess an atypical DNA content. Therefore, we postulate that functional vesicular trafficking is crucial for sustaining integral genome. We believe that numerous new genes implicated in genome maintenance after genotoxic oxidative stress, together with newly discovered vesicular trafficking link to genome integrity, will help revealing novel molecular processes involved in the genome stability of diploid cells. Screen for diploid Saccharomyces cerevisiae deletion strains sensitive to zeocin (the compound generating double strand breaks in DNA). The pool of strains from Yeast knock-out (YKO) collection (Open Biosystems) containing homozygous diploid strains bearing double deletion of non-essential genes and heterozygous diploid strains bearing single deletion of essential genes were exposed to treatment with either 5 or 15 µg/ml zeocin for 1 hour. The change in relative abundance of individual deletion strains in a pool, as a measure of their increased zeocin sensitivity was assessed by comparison to mock treated strain pool using barcode microarrays with two-color hybridization. Four biological replicates of mock-treated vs zeocin treated strains were performed, two of them using the pool of original YKO diploid strains, the other two with the derivative pool, in which all strains bore additional single can1Δ mutation so they were heterozygous at CAN1 locus (Alabrudzinska et al., 2011 PLoS ONE, 6, e21124). Each biological replicate had two technical replicates with dye swapping. A total of 16 individual hybridizations were made. With each hybridization both barcodes: UP-Tag and DOWN-Tag, were analyzed and for technical reasons microarray images for UP-Tag and DOWN-Tag probe sets had to be scanned and quantified independently so there are 32 raw data files associated with this experiment.