Spontaneous Mutations in Saccharomyces cerevisiae mtDNA Increase Cell-to-Cell Variation in mtDNA Amount

In a eukaryotic cell, the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) is usually maintained within a specific range. This suggests the presence of a negative feedback loop mechanism preventing extensive mtDNA replication and depletion. However, the experimental data on this hypothetical mechanism are limited. In this study, we suggested that deletions in mtDNA, known to increase mtDNA abundance, can disrupt this mechanism, and thus, increase cell-to-cell variance in the mtDNA copy numbers. To test this, we generated Saccharomyces cerevisiae rho strains with large deletions in the mtDNA and rho strains depleted of mtDNA. Given that mtDNA contributes to the total DNA content of exponentially growing yeast cells, we showed that it can be quantified in individual cells by flow cytometry using the DNA-intercalating fluorescent dye SYTOX green. We found that the rho mutations increased both the levels and cell-to-cell heterogeneity in the total DNA content of G1 and G2/M yeast cells, with no association with the cell size. Furthermore, the depletion of mtDNA in both the rho and rho strains significantly decreased the SYTOX green signal variance. The high cell-to-cell heterogeneity of the mtDNA amount in the rho strains suggests that mtDNA copy number regulation relies on full-length mtDNA, whereas the rho mtDNAs partially escape this regulation.