dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

Significance An increased rate of mutation, or “mutator phenotype,” generates genetic diversity that can accelerate cancer progression or confer resistance to chemotherapy drugs. New therapeutic strategies are needed that target mutator phenotypes directly. Mutator phenotypes due to defects in DNA polymerase ε have been implicated in colorectal and endometrial cancers and may emerge in other cancers during treatment. Here, we show in budding yeast that such mutator phenotypes are influenced by the levels of dNTPs, the building blocks of DNA. Lowering dNTP pool levels lessens the mutator phenotypes, whereas increasing dNTP pools accentuates the mutator phenotypes. These findings suggest that mutator phenotypes due to error-prone polymerases may be modulated by treatments that target dNTP pools. Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 ( dun1 Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint ( MRC ) 1 ( mrc1 Δ) and radiation sensitive ( Rad ) 9 ( rad9 Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1 Δ but not rad9 Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1 Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1 Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthes