Topoisomerase 1 provokes the formation of short deletions in repeated sequences upon high transcription in Saccharomyces cerevisiae

Abstract

High transcription is associated with genetic instability, notably increased spontaneous mutation rates, which is a phenomenon termed Transcription-Associated-Mutagenesis (TAM). In this study, we investigated TAM using the chromosomal CAN1 gene under the transcriptional control of two strong and inducible promoters (pGAL1 and pTET) in Saccharomyces cerevisiae. Both pTET- and pGAL1-driven high transcription at the CAN1 gene result in enhanced spontaneous mutation rates. Comparison of both promoters reveals differences in the type of mutagenesis, except for short (-2 and -3 nt) deletions, which depend only on the level of transcription. This mutation type, characteristic of TAM, is sequence dependent, occurring prefentially at di- and trinucleotides repeats, notably at two mutational hotspots encompassing the same 5'-ACATAT-3' sequence. To explore the mechanisms underlying the formation of short deletions in the course of TAM, we have determined Can(R) mutation spectra in yeast mutants affected in DNA metabolism. We identified topoisomerase 1-deficient strains (top1Δ) that specifically abolish the formation of short deletions under high transcription. The rate of the formation of (-2/-3nt) deletions is also reduced in the absence of RAD1 and MUS81 genes, involved in the repair of Top1p-DNA covalent complex. Furthermore ChIP analysis reveals an enrichment of trapped Top1p in the CAN1 ORF under high transcription. We propose a model, in which the repair of trapped Top1p-DNA complexes provokes the formation of short deletion in S. cerevisiae. This study reveals unavoidable conflicts between Top1p and the transcriptional machinery and their potential impact on genome stability.