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

Abstract

The URA3 gene is frequently used in the budding yeast community as the mutation target for 5-fluoroorotic acid (5-FOA) resistance. We identified a class of ura6 mutants that can grow in the presence of 5-FOA. Unlike ura3 mutants, ura6 mutants remain prototrophic and are able to grow in the absence of uracil. In addition to 5-FOA resistance, we found that ura6 mutants are also resistant to 5-fluorocytosine (5-FC) and 5-fluorouracil (5-FU). In total, we identified 50 unique missense mutations across 32 unique amino acid positions of Ura6 which confer resistance to 5-FOA. We found that 28 out of the 32 affected positions are located in regions conserved between Saccharomyces cerevisiae and three clinically relevant pathogenic fungi. Metabolic analysis revealed a build-up of uridine monophosphate (UMP) and 5-fluorouridine monophosphate (5-FUMP) in ura6 mutants, indicating a reduction in Ura6 activity. Despite the accumulation of UMP in ura6 mutants, uridine diphosphate and triphosphate (UDP, UTP) levels were similar across mutants and wild type. These findings suggest that missense mutations to URA6, can result in reduction of uridylate kinase activity and lead to cross resistance to fluorinated prodrugs that incorporate into both the de novo pyrimidine synthesis and the pyrimidine salvage pathway.