Investigating and Characterizing Half Crossover Cascades in Saccharomyces cerevisiae
Category: Research Poster
Author(s): Via Lawson
Presenter(s): Via Lawson
Mentors(s): Juan Argueso, Ruth Watson
We are examining the rate at which a specific genetic event, called a Half Crossover Cascade (HCC), occurs when normal genome maintenance is compromised. An HCC occurs when DNA is broken on one chromosome outside a homologous sequence and uses a homologous sequence on another chromosome to attempt to repair. The broken chromosome will have a ssDNA piece, that is clipped by a protein called Rad1. We wanted to determine if this removal of the ssDNA tail is required for HCC to proceed. To determine this, we engineered 2 kb homology regions on three chromosomes (Chr6, Chr9, and Chr10) in the Saccharomyces cerevisiae genome, then used CRISPR/Cas9 to induce single-ended DSBs four and ninety bases away from the homology sequence on Chr6, thus shunting cells toward HCCs. Cells carrying the ensuing chromosomal translocations were positively identified through the restoration of an auxotrophic gene (LYS2). Clones recovered through this assay were then quantified then phenotypically characterized by patching onto various media for auxotrophic and drug resistance markers. We examined changes in the frequency of Lys+ formation and the mutational spectra of Lys+ colonies when the RAD1 gene is knocked out as compared to WT. In clones where the break occurred 90 bases away from the homologous sequence we observed a significant decrease in rad1Δ clones which survived and produced HCC as opposed to the WT. In contrast, when the break occurred 4 bases away from the homologous sequence, we found there was not a significant decrease in Lys+ frequency.