Uridine-sensitized screening identifies genes and metabolic regulators of nucleotide synthesis.

Nucleotides are essential for nucleic acid synthesis, signaling, and metabolism, and can be synthesized de novo or through salvage. Rapidly proliferating cells require large amounts of nucleotides, making nucleotide metabolism a widely exploited target for cancer therapy. However, resistance frequently emerges, highlighting the need for a deeper understanding of nucleotide regulation. Here, we harness uridine salvage and CRISPR-Cas9 screening to reveal regulators of de novo pyrimidine synthesis. We identify several factors and report that pyrimidine synthesis can continue in the absence of coenzyme Q (CoQ), the canonical electron acceptor in de novo synthesis. We further investigate NUDT5 and report its conserved interaction with PPAT, the rate-limiting enzyme in purine synthesis. We show that in the absence of NUDT5, hyperactive purine synthesis siphons the phosphoribosyl pyrophosphate (PRPP) pool at the expense of pyrimidine synthesis, promoting resistance to chemotherapy. Intriguingly, the interaction between NUDT5 and PPAT appears to be disrupted by PRPP, highlighting intricate allosteric regulation. Our findings reveal a fundamental mechanism for maintaining nucleotide balance and position NUDT5 as a potential biomarker for predicting resistance to chemotherapy.