Role for topoisomerase 1 in transcription-associated mutagenesis in yeast

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 2; pp. 698 - 703
• Main Authors: Lippert, Malcolm J., Kim, Nayun, Cho, Jang-Eun, Larson, Ryan P., Schoenly, Nathan E., O'Shea, Shannon H., Jinks-Robertson, Sue, Haber, James E.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 11.01.2011; National Acad Sciences
• Subjects: Alleles; Base Sequence; Biological Sciences; CAN1 gene; Conservation; DNA; DNA - genetics; DNA Damage; DNA Mutational Analysis; DNA repair; DNA Topoisomerases, Type I - genetics; Enzymes; Frameshift Mutation; Fungal Proteins - genetics; Gene Deletion; Genes; Genetic mutation; hot spots; Lesions; Microsatellite repeats; Models, Genetic; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Open reading frames; Reversion; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Signatures; top1 gene; Transcription; Transcription, Genetic; Yeast; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1012363108

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.