Ribonucleotides are the most abundant non-canonical component of yeast genomic DNA and their persistence is associated with a distinctive mutation signature characterized by deletion of a single repeat unit from a short tandem repeat. These deletion events are dependent on DNA topoisomerase I (Top1) and are initiated by Top1 incision at the relevant ribonucleotide 3′-phosphodiester. A requirement for the re-ligation activity of Top1 led us to propose a sequential cleavage model for Top1-dependent mutagenesis at ribonucleotides. Here, we test key features of this model via parallel in vitro and in vivo analyses. We find that the distance between two Top1 cleavage sites determines the deletion size and that this distance is inversely related to the deletion frequency. Following the creation of a gap by two Top1 cleavage events, the tandem repeat provides complementarity that promotes realignment to a nick and subsequent Top1-mediated ligation. Complementarity downstream of the gap promotes deletion formation more effectively than does complementarity upstream of the gap, consistent with constraints to realignment of the strand to which Top1 is covalently bound. Our data fortify sequential Top1 cleavage as the mechanism for ribonucleotide-dependent deletions and provide new insight into the component steps of this process
