Role for topoisomerase 1 in transcription-associated mutagenesis in yeast

Abstract

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.

Fig. 1.

Short (2- to 5-bp) deletions in the pGAL-CAN1 spectrum. The entire CAN1 ORF is shown, and deletions are indicated above the relevant sequence as bars. The size of individual bars reflects the size of the corresponding deletion. Deletions isolated from WT, rad51Δ, and rev3Δ backgrounds are in charcoal, blue, and red, respectively. All dinucleotide repeats are highlighted in gray.

Fig. 2.

Reversion rates of lys2ΔA746NR alleles under high- and low-transcription conditions. LYS2 and TET promoters were used for low- and high-transcription conditions, respectively. White bars correspond to 2-bp deletions in the native LYS2 sequence, black bars to 2-bp deletions at the introduced (AT)₂ or (TC)₃ hotspot, and gray bars to all other mutations. The rate of all 2-bp deletions relative to that in the WT, low-transcription strain is indicated to the right of the graphs.

Fig. 3.

Model for Top1-dependent deletions. Sequence surrounding the (AT)₂ hotspot is shown. A possible Top1 consensus sequence is highlighted in gray, and the ends generated by Top1 cleavage are as indicated. See Discussion for additional details.