REV1 and REV7, but not RAD30, are required to observe increases in CAN1 mutation rate during replicative senescence in est2 mutant cells. (A) CAN1 mutation rate of (▴) wild type, (▵) est2Δ, ▪ rev1Δ, (•) rev7Δ, (▾) rad30Δ, and (♦) exo1Δ mutant cells at the indicated time points. (B) CAN1 mutation rate of (□) est2Δ rev1Δ, (○) est2Δ rev7Δ, (▿) est2Δ rad30Δ, (⋄) est2Δ exo1Δ, (×) rad30Δ rev7Δ, and (*) est2Δ rad30Δ rev7Δ mutant cells at the indicated time points. Spore colonies of the appropriate genotype were obtained from freshly dissected tetrads of ABX1269, ABX1727, and ABX1729 (Table 1) and dispersed in water. Aliquots were removed to determine viability following dilution, plating on to YPD medium, and incubation for 3 days at 30°. The remainder was plated onto synthetic medium lacking arginine and containing 60 μg/ml canavanine, and incubated for 3 days at 30°. The colonies arising on the canavanine plates were counted and replica plated to synthetic medium lacking uracil and the numbers of Ura⁻ and Ura⁺ colonies were determined after overnight incubation at 30°. CAN1 mutation frequency was determined by dividing the number of Can^r Ura⁺ colonies by the number of viable cells plated for each spore colony. CAN1 mutation rate was determined using the median CAN1 mutation frequency from at least 10 independent trials (Lea and Coulson 1949). Statistical significance was tested by determining the number of trials with each strain that were above and below the group median frequency and then performing χ²-analysis and Fisher's exact test. This process was repeated at five successive growth intervals ∼25 generations apart, using single colonies that arose on the YPD viability plates.

CYH2 mutation rate does not increase in telomerase-deficient cells during replicative senescence. CYH2 mutation rate of (♦) wild type and (⋄) est2Δ mutants was determined at the indicated time points. Wild-type and est2 mutant spore colonies were obtained from freshly dissected tetrads of ABX1429 (Table 1) and dispersed in water. Aliquots were removed to determine viability following dilution, plating on to YPD medium, and incubation for 3 days at 30°. The remainder was plated onto synthetic medium containing 1 μg/ml cycloheximide and incubated for 3 days at 30°. CYH2 mutation frequency was determined by dividing the number of Cyh^r colonies by the number of viable cells plated for each spore colony. CYH2 mutation rate was determined using the median CYH2 mutation frequency from at least 10 independent trials (Lea and Coulson 1949). Statistical significance was tested by determining the number of trials with each strain that were above and below the group median frequency and then performing χ²-analysis and Fisher's exact test. This process was repeated at two additional growth intervals ∼25 generations apart, using single colonies that arose on the YPD viability plates.

Rev1, Rev7, and Rad30 do not significantly affect senescence and subsequent recovery. (A) (▴) Wild type, (•) rev7Δ, (▪) rev1Δ, and (▾) rad30Δ. (B) (▵) est2Δ, (○) est2Δ rev7Δ, (□) est2Δ rev1Δ, (▿) est2Δ rad30Δ, (×) rad30Δ rev7Δ, and (*) est2Δ rad30Δ rev7Δ. Serial liquid growth was performed as described previously (Hackett et al. 2001). During each day of serial liquid growth, hemocytometer counts were performed to determine the number of cell bodies, after which ∼500 cells were plated to YPD and incubated at 30° for 3 days. Colonies were then counted and divided by 500 to determine plating efficiency. Finally, viability was determined following each day of growth in liquid culture by taking the number of cell bodies and multiplying by the plating efficiency. Results are the mean ±2 SE from at least eight independent samples of each indicated genotype.