URA6 mutations provide an alternative mechanism for 5-FOA resistance in Saccharomyces cerevisiae

Abstract

URA3 is frequently used in the yeast community as the mutation target for 5-fluoroorotic acid (5-FOA) resistance. We identified a novel class of ura6 mutants that can grow in the presence of 5-FOA. Unlike ura3 mutants, ura6 mutants remain prototrophic and grow in the absence of uracil. In addition to 5-FOA resistance, we found that mutations to URA6 also confer resistance to 5-fluorocytosine (5-FC) and 5-fluorouracil (5-FU). In total, we identified 50 unique missense mutations across 32 residues of URA6. We found that 28 out of the 32 affected residues are located in regions conserved between Saccharomyces cerevisiae and three clinically relevant pathogenic fungi. These findings suggest that mutations to URA6 present a second target for mutation screens utilizing 5-FOA as a selection marker as well as a potential mode of resistance to the antifungal therapeutic 5-FC.

Figure 1.

Growth of ura6 mutants in media containing toxic fluorinated pyrimidine analogs. A) Summary comparison of qualitative growth phenotypes across WT, ura3, and ura6 mutants in YPD, SC-ura, 5-FOA, SC-ura +5-FOA. “+” indicates growth, and “−” indicates lack of growth. B) URA6 lies at the junction of the pyrimidine synthesis and salvage pathways. The fluorinated drugs 5-FC and 5-FU are incorporated into the pyrimidine salvage pathway. 5-FOA is incorporated into the pyrimidine synthesis pathway. The production of 5-FdUMP and 5-FUTP from the fluorinated prodrugs leads to toxicity. C) Growth curves for three biological replicates of ura6 mutants and wild-type in MM, MM+5-FOA, MM+5FC, MM+5FU measured at 30°C. D) Area under the growth curve (AUC) measurements for growth for ura6 mutants and wild-type strains grown in MM, MM+5-FOA, MM+5FC, MM+5FU measured at 30°C. E) Growth curves for three biological replicates of individual ura6 mutants and wild type strains grown in MM at 30°C and 37°C.

Figure 2.. 5-FOA resistance mutations occur in evolutionarily conserved regions of URA6.

White circles denote the missense Ura6 mutations identified in this study which confer resistance to fluorinated pyrimidine derivatives. Annotated binding sites are shown in green (ATP) and in gray (ribonucleotide 5’-phosphate). Conserved Ura6 residues between 1,011 natural S. cerevisiae isolates are shown in blue. Conserved residues between S. cerevisiae (S288C), A. fumigatus (Af293), C. neoformans (JEC21), and C. albicans (SC5314) are shown in yellow.

Figure 3.. Working model for ura6 mutants fluorinated prodrug resistance.

A) The de novo pyrimidine synthesis and salvage pathway in the WT cells. The production of F-UTP and F-CTP leads to cell death. B) The de novo pyrimidine synthesis and salvage pathway in ura6 mutant cells at 30°C. We propose that missense mutations can reduce Ura6 activity, leading to the build-up of UMP in the cells. A reduction in UMP kinase activity could enable these mutants to generate enough UTP and CTP for the cells to survive, but avoid producing enough F-UTP and F-CTP to lead to cell death.