Figure 4.

The peroxidatic cysteine in Tsa1 is required to protect against H₂O₂ and lower mutation rates. (A) Western blot analysis of FLAG-tagged Tsa1 variants. Pgk1 levels were monitored as a loading control. (B) Stationary phase cultures of wild-type (BY4741) cells transformed with vector, tsa1Δ tsa2Δ cells transformed with vector, or tsa1Δ tsa2Δ cells expressing FLAG-tagged Tsa1 variants were diluted serially, plated on non-selective growth medium (YPD) or YPD containing 4 mM H₂O₂, and grown for 48 h at 30 °C. Results are representative of three independent experiments. (C) Mutation rates in cells expressing Tsa1 variants were determined by monitoring fluctuation of the CAN1 gene, as assessed by counting canavanine-resistant colonies for nine independent isolates of each strain in duplicate. The graph depicts the median mutation rate ± the 95% confidence limit for each strain. Detailed methods are available in Supplementary Materials.