Topoisomerase I promotes formation of a novel DNA intermediate in the ribosomal DNA

Abstract

The ribosomal DNA (rDNA) is a conserved but highly unstable multi-gene locus that codes for the RNA components of the ribosome (rRNA). Despite being essential for protein translation and survival, rRNA gene-copy numbers fluctuate frequently. Here, we describe a novel rDNA intermediate that may be involved in these copy-number changes in Saccharomyces cerevisiae. Two-dimensional gel analyses revealed a structure that includes a large single-stranded tail and arises from the promoter of the 35S rRNA gene. Formation of this intermediate is unaffected by reduced 35S transcription but relies on topoisomerase I (Top1). Unexpectedly, intermediate formation is also independent of S phase or the rDNA-instability factor Fob1, even though Fob1 is mediates the major peak of Top1 cleavage complexes in the rDNA. Indeed, we find that the known 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 also largely independent of Fob1. We therefore speculate that failure to form this intermediate leads to rDNA instability in top1 mutants.