Detection and Manipulation of Extrachromosomal Circular DNA in Yeast

Extrachromosomal circular DNA (eccDNA) has become an increasingly popular subject of study in eukaryotic cell biology due to its prevalence in human cancer. Though the literature reports a consensus regarding DNA break repair as a driver of eccDNA formation, there remains a lack of knowledge surrounding the exact mechanisms for eccDNA formation and the selective dynamics that promote their retainment in a cell or population. A central issue to studying eccDNA is the inability to distinguish between linear and circular DNA of homologous sequence. The work presented here describes an adapted eccDNA enrichment and detection assay, specifically for investigating the effects of manipulating a known eccDNA-forming locus in the budding yeast Saccharomyces cerevisiae. First, a galactose inducible GFP reporter was integrated within the copper inducible CUP1 tandem repeat locus of yeast cells. The eccDNA enrichment and detection assay was first applied to wildtype yeast to demonstrate the presence of CUP1 eccDNA in copper induced cells by qPCR. Although subsequent sequencing analysis failed to validate this result, it indicated the presence of various other known and previously un-reported eccDNA species. Finally, application of the enrichment protocol and qPCR detection assay to CUP1-GFP reporter cells yielded inconclusive results, suggesting the assay requires further optimization to sensitively detect eccDNA from this altered locus. While more work is necessary to draw conclusions regarding the limits of eccDNA production at a manipulated eccDNA-forming locus, this knowledge will lend to the potential for therapeutically targeting eccDNA at the point of de novo formation.