| DNA polymerases (Pols) delta and epsilon perform the bulk of eukaryotic nuclear DNA replication. Both Pols are high-fidelity enzymes with intrinsic proofreading exonucleases that remove errors during DNA synthesis. Errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Cells that lack Pol proofreading or MMR exhibit a mutator phenotype, manifested as a 10- to 100-fold increase in spontaneous mutation rate. Haploid yeast strains with combined defects in Pol proofreading and MMR initially divide, but succumb to error-induced extinction and fail to form viable colonies. We exploited error-induced extinction to isolate suppressors of lethal mutation rates (eex mutants). In screens for mutants that suppress pol3-01 msh6-delta or pol2-4 msh2-delta error-induced extinction, thirty-five- and six-percent of eex mutations, respectively, encoded 'antimutator' polymerases that increased replication fidelity. The locations of antimutator amino-acid changes and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. The remaining eex alleles were extragenic to the polymerase genes, suggesting that factors in addition to polymerase base selectivity, proofreading and MMR influence replication fidelity. Previous studies (Datta et al. 2000) showed that the mutator phenotype of proofreading-deficient Pol delta depends on Dun1, a protein kinase that mediates damage-inducible gene expression and up-regulates dNTP synthesis during the S-phase checkpoint. We demonstrated that deletion of Dun1 (dun1-delta) rescues cells from error-induced extinction caused by combined defects in Pol epsilon proofreading and base selectivity (pol2-4,M644G) or Pol epsilon proofreading and mismatch repair (pol2-4 msh2-delta). dun1-delta suppressed the mutator phenotypes of pol2-4 (encoding proofreading-deficient Pol epsilon) and pol2-M644G (encoding a Pol epsilon variant with altered base selectivity) 7- to 10-fold. Our findings, together with those of Datta et al., suggest that Pol delta and Pol epsilon errors are sensed as stressors that trigger Dun1 activation. Our data present a complex picture of DNA replication fidelity, where misincorporations during replication trigger Dun1-dependent mutagenesis. We propose that Dun1 stimulation of dNTP synthesis enhances mispair extension to ensure continued DNA replication. Thus, it appears that mutator phenotypes are inducible and suppressible through multiple mechanisms. We speculate that transient mutator phenotypes promote microbial adaptation and mammalian oncogenesis. |
