High levels of transcription in Saccharomyces cerevisiae are associated with 
increased genetic instability, which has been linked to DNA damage. Here, we 
describe a pGAL-CAN1 forward mutation assay for studying 
transcription-associated mutagenesis (TAM) in yeast. In a wild-type background 
with no alterations in DNA repair capacity, ≈50% of forward mutations that arise 
in the CAN1 gene under high-transcription conditions are deletions of 2-5 bp. 
Furthermore, the deletions characteristic of TAM localize to discrete hotspots 
that coincide with 2-4 copies of a tandem repeat. Although the signature 
deletions of TAM are not affected by the loss of error-free or error-prone 
lesion bypass pathways, they are completely eliminated by deletion of the TOP1 
gene, which encodes the yeast type IB topoisomerase. Hotspots can be transposed 
into the context of a frameshift reversion assay, which is sensitive enough to 
detect Top1-dependent deletions even in the absence of high transcription. We 
suggest that the accumulation of Top1 cleavage complexes is related to the level 
of transcription and that their removal leads to the signature deletions. Given 
the high degree of conservation between DNA metabolic processes, the links 
established here among transcription, Top1, and mutagenesis are likely to extend 
beyond the yeast system.