Genome-wide approaches for identifying genes involved in the maintenance of genomic stability

The maintenance of genomic stability and the repair of DNA damage are essential for the survival of all cells. Despite diverse pathways for repair of DNA lesions, different mutations can arise, ranging from Single Nucleotide Variants (SNVs) to larger Structu- ral Variants (SVs). The processes that play a role in the formation of these alterations are not fully understood. In this thesis, I present two complementary approaches for accumulating genomic variants and for identifying pathways involved in the suppression of mutation formation using Saccharomyces cerevisiae (budding yeast) gene knockout strains. First, using next-generation sequencing, I studied neutral variants through a mutation accumulation assay for up to 1800 generations. I used 47 yeast strains with known defects in DNA replication, repair and recombination pathways. In all strains, small insertions and deletions (indels) were more common than larger SVs (>50bp). Most mutations occurred in repetitive sequences, implicating replication based mechanisms and homologous recombination in the formation of genomic variants. Furthermore, the knockout of MSH2 produced a hypermutable strain that acquired the highest number of indels. Moreover, the knockout of the genes SWR1 and ISW1, involved in chromatin remodeling, resulted in strains with high number of deletions. These results suggest that defects in establishing a correct chromatin architecture may play a role in the formation of genomic variants. I further performed a genome-wide screen for genes that suppress deletion formation under different drug treatments in the presence or absence of homologous repeats by using designed constructs. As expected, deletions occurred more often between repeats, in support of the frequent involvement of homologous recombination in the formation of chromosome rearrangements. In addition, I identified genes whose knockout led to incre- ased levels of deletions. Among these, IOC4 is of particular interest given that it belongs to the same chromatin remodeling complex as ISW1, identified in the neutral mutati- on accumulation assay. This provides further evidence that chromatin remodeling may be involved in preventing the occurrence of SVs. Furthermore, several meiosis-related mutants also showed increased levels of deletions, suggesting that meiosis proteins may have additional roles in the maintenance of genomic stability during vegetative growth. By performing additional experimental validations, I verified the higher vulnerability of meiosis gene knockouts to acquire deletions, especially in their diploid stages. In the last chapter, I briefly describe the results of several side projects in which I applied computational methods learned through the above mentioned projects, to identify and characterize genomic rearrangements in different human cancers. In summary, I have found that genome-wide approaches can provide interesting insights into the understanding of genomic variants in yeast and human cancers. In particular, given the evolutionary conservation of the ISWI chromatin remodeling complex and meiosis-related genes, the results presented here point to potentially novel functions of these proteins in the maintenance of genomic stability.