Evidence for a threshold of error-induced extinction.
A) Entry into error-induced extinction. Mutated pol3 alleles were introduced into haploid MSH6 and msh6Δ yeast by plasmid shuffling (Figure S1), and mutation rates were measured by fluctuation assays and calculated using the maximum likelihood method. Each bar represents the spontaneous mutation rate, expressed as canavanine-resistant (Canr) mutants per cell division, conferred by a specific POL3 allele in MSH6 or msh6Δ cells. Mutation rate values (x 10−7) are indicated on each column. Error bars show 95% confidence intervals. WT, wild-type POL3; black, MSH6; gray, msh6Δ; X, no growth. B) Synthetic lethality of strong pol3 mutator alleles with msh6Δ. Serial dilutions of haploid yeast containing POL3–URA3 and pol3–LEU2 plasmids were plated on SC FOA medium to select for cells that spontaneously lost POL3–URA3. Similar numbers of cells (∼105, 104 and 103) were plated for each set of alleles in the MSH6 and msh6Δ strains. Failed growth of msh6Δ cells carrying pol3-D407A or pol3-01 indicates synthetic lethality (right two panels). pol3-F406A and pol3-Y516F also failed to support colony formation in msh6Δ cells (not shown). Note the small size of pol3-D463A msh6Δ colonies.