Identification of a Distinctive Mutation Spectrum Associated with High Levels of Transcription in Yeast

Abstract

High levels of transcription are associated with increased mutation rates in Saccharomyces cerevisiae, a phenomenon termed transcription-associated mutation (TAM). To obtain insight into the mechanism of TAM, we obtained LYS2 forward mutation spectra under low- versus high-transcription conditions in which LYS2 was expressed from either the low-level pLYS2 promoter or the strong pGAL1-10 promoter, respectively. Because of the large size of the LYS2 locus, forward mutations first were mapped to specific LYS2 subregions, and then those mutations that occurred within a defined 736-bp target region were sequenced. In the low-transcription strain base substitutions comprised the majority (64%) of mutations, whereas short insertion-deletion mutations predominated (56%) in the high-transcription strain. Most notably, deletions of 2 nucleotides (nt) comprised 21% of the mutations in the high-transcription strain, and these events occurred predominantly at 5′-(G/C)AAA-3′ sites. No −2 events were present in the low-transcription spectrum, thus identifying 2-nt deletions as a unique mutational signature for TAM.

We thank Sarah Musella for technical assistance, Nayun Kim for quantifying RNA, Douglas Johnson (University of Vermont) for his gift of strain SLY186, Mark Longtine (Oklahoma State University) for his gift of plasmid pFA6a-kanMX6-PGAL1, and George Hoffman (College of the Holy Cross) for valuable discussions and helpful comments on the manuscript.

This work was supported by Vermont Experimental Program to Stimulate Competitive Research grant number EPS 0236976 to M. J. Lippert and the Vermont Genetics Network through National Institutes of Health grant number 1 P20 RR16462 from the BRIN Program of the National Center for Research Resources to M. J. Lippert. In addition, this work was supported by National Institutes of Health grant GM-38464 to S. Jinks-Robertson. J. A. Freedman was partially supported by the Graduate Division of Biological and Biomedical Sciences of Emory University. M. A. Barber was supported by a Summer Undergraduate Research Fellowship from Pfizer, Inc.