Analyzing Genomic Studies and a Screen for Genes that Suppress Information Loss During DNA Damage Repair

This thesis is concerned with the means by which cells preserve genetic information and, in particular, with the competition between different DNA damage responses. DNA is continuously damaged and imperfect repair can have extremely detrimental effects. Double strand breaks are the most severe form of damage and can be repaired in several different ways or countered by other cellular responses. DNA context is important; cell cycle, chromosomal structure, and sequence all can make DSBs more likely or more problematic to repair. Saccharomyces cerevisiae is very resilient to DSBs and primarily uses a process called homologous recombination to repair DNA damage. To further our understanding of how S. cerevisiae efficiently uses homologous recombination, and thereby minimizes genetic degradation, I performed a screen for genes affecting this process. >In devising this study, I set out to quickly quantify the contribution of every non-essential yeast gene to suppressing genetic rearrangements and deletions at a single locus. Before I began I did not fully appreciate how variable and contingent this type of recombination phenotype could be. Accounting for the complex and changing recombination baseline across many tests became a significant effort unto itself. The requirements of the experimental protocols precluded the use of traditional recombination rate calculation methods. Searching for the means to compare the utility of normalizations and to validate my results, I sought general approaches for analyzing genome wide screen data and coordinating interpretation with existing knowledge. It was advantageous during this study to develop novel analysis tools. The second chapter describes one of these tools we developed, a technique called CLIK (Cutoff Linked to Interaction Knowledge). CLIK uses preexisting biological information to evaluate screen performance and to empirically define a significance threshold. This technique was used to analyze the screen results described in chapter three. The screen in chapter three represents the primary work of this dissertation. Its purpose was to identify genes and biological processes important for the suppression of recombination between DNA tandem repeats in yeast. By searching for gene deletion strains that show an increase in non-conservative single strand annealing, I found that many genetic backgrounds could induce altered recombination frequencies, with genes involved in DNA repair, mitochondria structural and ribosomal, and chromatin remodeling genes being most important for minimizing the loss of genetic information by HR. In addition, I found that the remodeling complex INO80 subunits, ARP8 and IES5 are significant in suppressing SSA.