Genomic copy-number loss is rescued by self-limiting production of DNA circles, Mansisidor et al.
Description
The major goal of this work was to determine the relationship between copy number of the ribosomal RNA gene cluster (rDNA) and the production of extrachromosomal rDNA cirlces (ERCs). Using Saccharomyces cerevisiae, we developed a collection of strains with different sizes of the chromosomal rDNA array ranging from 90 - 180 repeats. These strains also allowed us to follow the fate of individual repeats by inserting a unique marker sequence within the rDNA array. A major finding of this work was that strains with smaller chromosomal rDNA array (90 repeats) produce more circles compared to strains with larger chromosomal rDNA arrays (180 repeats). Also, the data showed that the production of ERCs is not the formation of repeat loss from the chromosomal array, but instead occurs through a mechanism that amplifies repeat copy number. We further connected ERC copy number with rRNA levels and with re-insertion of ERC into the chromosomal array, suggesting that ERCs may play positive role physiologically to expand rDNA copy number. The underlying data is primarily quantitative Southern blots detecting ERCs from all repeats, as well as from individual marked repeats. Notably, we used a single-copy loading control (GAT1 or URA3) to compare ERC levels to instead of the traditionally used rDNA chromosomal array signal that can be misleading given the anti-correlation we have discovered.