FIG 3.

(A) The upp gene encodes UPRTase, which is a key enzyme in the uracil salvage pathway. The product of the UPRTase reaction, UMP, is processed by downstream enzymes in pathways for RNA, DNA, and sugar nucleotide synthesis. 5-FU causes cell death by incorporating into this pathway via UPRTase. (B) Schematic of genomic regions of upp in the WT or the ΔMAI mutant (top) and the Δupp mutant (bottom). (C) Genomic region of kup in WT (top) and kup::strAB (bottom) strains. Primers used to screen for the correct genotype are indicated with half arrows. (D) Δupp mutants in WT and ΔMAI backgrounds were confirmed by PCR using primers P19/P20 and agarose gel electrophoresis. WT and ΔMAI strains show a band corresponding to the upp gene (2,691 bp), while the Δupp mutants have a smaller band corresponding to a markerless deletion of the upp gene (2,079 bp). The lower bands are likely nonspecific PCR products. (E) kupM::strAB genotype confirmation by PCR and agarose gel electrophoresis using primers P21/P22 (WT, 3,069 bp; kupM::strAB, 3,263 bp; ΔMAI, not applicable [NA]).