S. cerevisiae strains were grown in YPD (1% yeast extract, 2% Bacto Peptone and 2% dextrose) or in the appropriate synthetic dropout media (0.67% yeast nitrogen base without amino acids, 2% dextrose, and amino acid dropout mix at the concentration recommended by the manufacturer (US Biological) at 30 °C. All S. cerevisiae strains in this study were derived in the S288c strain background using standard gene deletion and pop-in, pop-out methods.
Mutator phenotypes were evaluated using the hom3-10 frameshift reversion assay. Mutation rates were determined by fluctuation analysis using a minimum of 2 independently derived strains and 14 or more independent cultures; comparisons of mutation rates were evaluated using 95% confidence intervals.
One independent Thr+ revertant was isolated per culture from fluctuation tests. Chromosomal DNA was isolated from each revertant using a Qiagen Puregene Yeast/Bact. Kit B and the hom3-10 region were amplified by PCR using the Primer 5′-AGTTGTTTGTTGATGACTGC and Primer 5′-TTCAGAAGCTTCTTCTGGAG and sequenced with the Primer 5′-CTTTCCTGGTTCAAGCATTG using a commercial sequencing facility (Calil et al., 2021 (link)).
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