GoKawiilData reporting
No statistical methods were used to predetermine sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.
Mammalian cell culture
U2OS and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax (Gibco, 10566016), and MOLT4, Jurkat and NALM6 cells were maintained in RPMI-1640 medium with glutamine (Gibco, 11875093). The medium was supplemented with 10% fetal bovine serum (FBS; VWR, 89510-186) and 1× penicillin–streptomycin (Pen-Strep; Gibco, 15140122) at 37 °C and 5% CO 2 . The growth medium for PPAT-knockout cells was supplemented with 100 µM adenine. For drug treatment immunoprecipitation experiments, cells were plated in DMEM with Glutamax, 10% dialysed FBS (dFBS; Cytiva, SH30079.02) and 1× Pen-Strep. For metabolomics experiments, cells were plated in DMEM (high glucose, no glutamine; Gibco, 11960044) supplemented with 2 mM l-glutamine (Gibco, A2916801), 10% dFBS and 1x Pen-Strep. Further details about cell culture conditions for isotopic tracing experiments can be found under ‘Mass-spectrometry-based metabolomics’. Expi293F cells were maintained in Epxi293 medium (Gibco; A1435101) in shaker flasks at 130 rpm and 8% CO 2 . Cell stocks were obtained from and authenticated by (using short tandem repeat profiling) the University of California Cell Culture Facility (RRID: SCR_017924) and were routinely tested for mycoplasma contamination.
CRISPR editing and endogenous cell line generation
To generate CRISPR knockout NUDT5 and PPAT lines in HEK293T, Expi293F, MOLT4, Jurkat and NALM6 cells, we used a mixture of three optimized sgRNAs (Gene Knockout Kit v.2, Synthego). A total of 1.5 μl sgRNA mixture (100 μM) was incubated with 3.2 μl Cas9-NLS protein (40 μM; produced by the University of California, Berkeley (UC Berkeley) MacroLab) for 10 min at room temperature. Ribonucleoproteins (RNPs) were nucleofected into 5 × 105 cells resuspended in Lonza SF solution (Lonza, V4XC-2032) using a Lonza 4D Nucleofector X-unit and pulse code CM-130 (HEK293T and Expi293F), CA-137 (MOLT4) or CM-138 (NALM6). Jurkat cells were nucleofected in Lonza SE solution (Lonza, V4XC-1032) using pulse code CL-120. Cells were recovered for 3-4 days, split once and then cloned by single-cell dilution in 96-well plates. Expi293F cells were not cloned, because NUDT5 bulk depletion was observed to be more than 95% by western blot. Clones were assessed for knockout by western blotting and phenotypic assay. Uncropped western blots and gels are included as Supplementary Fig. 1. To establish NUDT5-knockout lines used for experiments, five clones were combined at equal cell number into one pool to minimize clonal bias effects. A similar editing strategy was used for endogenous 3×Flag epitope tagging and NUDT5L217A/K218A mutant generation, except that a single sgRNA was used and 1 μl Alt-R HDR single-stranded oligodeoxynucleotide (ssODN) donor template (100 μM stock; IDT) was included in the nucleofection mixture added after RNP formation. For knock-in generation, cells were recovered in HDR Enhancer v.2 (IDT) for 16 h after nucleofection, followed by a change of medium. Knock-in clones were validated by Sanger sequencing. The sgRNA and ssODN sequences used are listed below:
sgRNA sequences: NUDT5 gene knockout (gKO) 1: 5′-CTTGCAGGTCTCATAGATGA-3′; NUDT5 gKO 2: 5′-ATCAATCCCTTCCCAGACGG-3′; NUDT5 gKO 3: 5′-ATACCAACCTGGAGAACATT-3′; PPAT gKO 1: 5′-TGATAGCAGTAGGACAATAA-3′; PPAT gKO 2: 5′-GTGTCTGATATAAATGACAA-3′; PPAT gKO 3: 5′-CATCTCTGTGCATTATAAGC-3′; NUDT5 endo-3×Flag: 5′-AATGGAGAGCCAAGAACCAA-3′; PPAT endo-3×Flag: 5′-TGTAGAATTAGAATGGTAGC-3′; NUDT5L217A/K218A: 5′-TGGGCTTAAAATTTCAAGAA-3′.
ssODN sequences: 3×FlagNUDT5: 5′-AACTTCTCACCTGAGGGCTGTAAAGACTCGTTTGAAAATGGACTACAAGGACCACGACGGCGATTATAAGGATCACGACATCGACTACAAAGACGACGATGACAAGGGTGGGAGTGGCGGGAGTGAGAGCCAAGAACCAACGGAATCTTCTCAGAATGGCAAACAGTATATCATTTC-3′; PPAT3×Flag: 5′-AGCTTGTCTCACTGGAAAATATCCTGTAGAATTAGAATGGGGTGGCAGCGGCGGTTCTGGTGGCAGCGACTACAAGGACCACGACGGCGATTATAAGGATCACGACATCGACTACAAAGACGACGATGACAAGTAGCTGGTAGGGTTGGATGTGTGTAGTTTCAAGATAGAAAG-3′; NUDT5L217A/K218A: 5′-TCGTTTACAAAAATGGCCAGTGTCATATTTGGGCTTAAAATGCAGCGAAGGGCACTTCAAATGGCTTTGCATTTGCATGTTTCAGT-3′.
Stable cell line generation
Lentiviral particles were generated by co-transfecting pLVX-IRES-puromycin or blasticidin constructs encoding the desired protein mutants with second-generation lentiviral packaging plasmids into HEK293T cells using Mirus Trans-IT LT1 transfection reagent (Mirus, MIR 2304). For FACS-based growth competition experiments, pLVX-mCherry-P2A-blasticidin and pLVX-GFP-P2A-blasticidin constructs were used to establish fluorescent cell lines for co-culture. After transfection of the plasmid mixture, the medium was changed the next day, and virus was collected 48 h after the medium change by filtering through a 0.45-µm filter. For stable cell line generation, cells were infected with lentiviral particles at a multiplicity of infection (MOI) < 1 in medium supplemented with polybrene (8 μg ml−1). Cells were selected with antibiotic (1 μg ml−1 puromycin or 10 μg ml−1 blasticidin) around 48 h after transduction until all cells on a control plate were dead, at which point selected cells were recovered without antibiotics for at least 2 days before assaying.
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