dUTPase and uracil-DNA glycosylase are central modulators of antifolate toxicity in Saccharomyces cerevisiae

The thymidylate synthase reaction remains an important target for widely used anticancer agents; however, the clinical utility of these drugs is limited by the occurrence of cellular resistance. Despite the considerable amount of information available regarding mechanisms of drug action, the relative significance of downstream events that result in lethality remains unclear. In this study, we have developed a model system using the budding yeast Saccharomyces cerevisiae to dissect the influence of dUMP misincorporation into DNA as a contributing mechanism of cytotoxicity induced by antifolate agents. The activities of dUTPase and uracil-DNA glycosylase, key enzymes in uracil-DNA metabolism, were diminished or augmented, and the manipulated strains were analyzed for biochemical endpoints of toxicity. Cells overexpressing dUTPase were protected from cytotoxicity by their ability to prevent dUTP pool expansion and were able to recover from an early S-phase checkpoint arrest. In contrast, depletion of dUTPase activity leads to the accumulation of dUTP pools and enhanced sensitivity to antifolates. These cells were also arrested in early S-phase and were unable to complete DNA replication after drug withdrawal, resulting in lethality. Inactivation of uracil base excision repair induced partial resistance to early cytotoxicity (within 10 h); however, lethality ultimately resulted at later time points (12-24 h), presumably because of the detrimental effects of stable uracil misincorporation. Although these cells were able to complete replication with uracil-substituted DNA, they arrested at the G(2)-M phase. This finding may represent a novel mechanism by which the G(2)-M checkpoint is signaled by the presence of uracil-substituted DNA. Together these data provide both genetic and biochemical evidence demonstrating that lethality from antifolates in yeast is primarily dependent on uracil misincorporation into DNA, and that uracil-independent mechanisms associated with dTTP depletion play a minor role. Our findings indicate that the relative expression levels of both dUTPase and uracil-DNA glycosylase can have great influence over the efficacy of thymidylate synthase-directed chemotherapy, thereby enhancing the candidacy of these proteins as prognostic markers and alternative targets for therapeutic development.