Monitoring DNA double strand break repair in Saccharomyces cerevisiae at high throughput and high resolution

The yeast mating type switching process is used as a biological model system to study eukaryotic DNA double strand break (DSB) repair, a process of fundamental importance in health and disease. However, tools for tracking the mating type switching process in real-time in live cells are limited. We developed methods to follow and quantify mating type switching occurrence in real-time, at the single live cell level, applicable for detection of rare events and population heterogeneity. First, we show, using a fluorescent reporter in live cells, that switching probability in strains tagged by a fluorescent repressor-operator system (FROS) is reduced, not only due to the integration of operator repeats, but also due to the overexpressed fluorescent molecules in the nucleus. Next, by applying wavefront shaping to imaging flow cytometry, we collected 3D data in high throughput, and were able to differentiate between MATa and MATα conformation and to detect DNA dynamics during switching with unprecedented spatial and temporal resolutions. Using a strain with the MAT-stk mutation, which gives rise to inefficient HO endonuclease DSB activity, we discriminated between two consecutive distances between the HMLα and the MAT loci upon DNA damage. We suggest that initial nearing between the loci occurs for both single and double strand breaks, and further and closer proximity occurs for DSB only, leading to mating type switching. Our approach can be adapted to other biological systems to study DSB repair with high precision, throughput and sensitivity.