Abstract
Topoisomerase I, a nickase that allows swiveling of the DNA substrate, is highly enriched in the ribosomal DNA (rDNA) from yeast to humans, but its function at this locus remains poorly understood. S. cerevisiae mutants lacking topoisomerase I (top1) exhibit pronounced rDNA instability and accumulate bubbles of single-stranded DNA (ssDNA) on 35S ribosomal RNA genes, suggesting a role in relieving transcription-associated topological stress. However, Top1 cleavage complexes are most highly enriched in the rDNA-encoded replication fork barrier, a genetically encoded source of rDNA instability, and only weakly in the 35S promoter, leading to the proposal that top1-associated rDNA instability may be linked to the fork barrier. Here, we show that the rDNA instability phenotypes of top1 mutants, including increased formation of extrachromosomal rDNA circles, elevated genetic marker loss, and instability of critically short rDNA arrays, are independent of the replication fork barrier. In addition, we link Top1 binding at the 35S promoter to the formation of a DNA species with a long ssDNA tail, which originates in the 35S promoter region and is undetectable in top1 mutants. This DNA species is abundant in wild-type cells, occurs independently of S-phase, and may be the resolution product of the ssDNA bubbles seen in top1 mutants. Whether the formation of this DNA species is important for rDNA stability remains unclear, but our findings link Top1 to highly active DNA metabolism in the 35S promoter.