High levels of transcription stimulate mutation rates in microorganisms, and this occurs primarily through an enhanced accumulation of DNA damage. The major source of transcription-associated damage in yeast is Topoisomerase I (Top1), an enzyme that removes torsional stress that accumulates when DNA strands are separated. Top1 relieves torsional stress by nicking and resealing one DNA strand, and some Top1-dependent mutations are due to trapping and processing of the covalent cleavage intermediate. Most, however, reflect enzyme incision at ribonucleotides, which are the most abundant noncanonical component of DNA. In either case, Top1 generates a distinctive mutation signature composed of short deletions in tandem repeats; in the specific case of ribonucleotide-initiated events, mutations reflect sequential cleavage by the enzyme. Top1-dependent mutations do not require highly activated transcription, but their levels are greatly increased by transcription, which partially reflects an interaction of Top1 with RNA polymerase. Recent studies have demonstrated that Top1-dependent mutations exhibit a strand bias, with the nature of the bias differing depending on the transcriptional status of the underlying DNA. Under low-transcription conditions, most Top1-dependent mutations arise in the context of replication and reflect incision at ribonucleotides incorporated during leading-strand synthesis. Under high-transcription conditions, most Top1-dependent events arise when the enzyme cleaves the non-transcribed strand of DNA. In addition to increasing genetic instability in growing cells, Top1 activity in transcriptionally active regions may be a source of mutations in quiescent cells.