Nuclear functions such as DNA damage repair are influenced by chromatin dynamics and are essential for maintaining genome integrity and stability across organisms. Among the most genotoxic lesions, DNA double-strand breaks (DSBs) are repaired through homologous recombination (HR). During HR, the DSB site and its homologous template undergo spatial reconfiguration, enabling their close proximity, which is required for the repair. The mating-type switching process in Saccharomyces cerevisiae provides a well-established model for studying DSB repair and chromatin dynamics. However, real-time tracking of mating-type switching in live cells remains limited by the resolution of existing techniques. Here, we developed high-throughput methods to monitor and quantify mating-type switching occurrences at the single live cell level, enabling the detection of rare events within heterogeneous populations. We revealed that switching probability in the labeled strains was reduced, both by the integration of operator repeats and by the presence of overexpressed nuclear fluorescent molecules. Next, by employing point spread function (PSF) engineering in an imaging flow cytometer, we visualized population-level loci dynamics in live cells and in 3D, which allowed us to distinguish between MATa and MATα conformations and to detect DNA conformational changes during switching with unprecedented resolution. In a MATa-stk mutant, which generates a mixed population of DSBs and single-strand breaks (SSBs) at the MAT locus, both lesion types promoted locus proximity, with tighter association observed for DSBs. These findings resolve ambiguities regarding chromatin dynamics during switching. Finally, using a PSF-engineered widefield microscope, we tracked DNA locus trajectories in 3D in live cells and monitored their dynamics over time. Together, these approaches provide a versatile platform for investigating the biophysical properties of DNA repair mechanisms across diverse systems, enabling high-throughput and precise analyses.
Ezra et al. (Sun,) studied this question.