Fig. 2: SIRT5 sustains the nucleotide pool by enhancing R5P synthesis to maintain DNA stability.

a Heatmap showing significantly differentially expressed metabolites in the PPP and purine/pyrimidine metabolism pathway after SIRT5 deletion in HCT116 cells. (n = 5 biologically independent experiments). b Targeted metabolomics analysis of nucleotides in HCT116 cells after SIRT5 knockdown. Data are expressed as mean ± standard error of the mean (SEM). Statistical significance was determined using the two-sided Student’s t test. (n = 6 biologically independent experiments). c–e Exogenous supplementation with nucleosides decreased the levels of γH2AX induced by SIRT5 silencing. Cells were transfected with SIRT5 siRNAs for 48 h and then cultured at indicated concentrations with four nucleosides (A, U, C, and G) for 16 h. Immunoblotting (c) and immunofluorescence staining (d) for γH2AX. Scale bars, 5 µm. Quantitation of the percentage of γ-H2AX-positive cells (e). (n = 3 biologically independent experiments). f, g Representative images of the alkaline comet assay for the SIRT5-knockdown HCT116 and LoVo cells under the indicated condition. Cells were cultured with four nucleosides (10 μM) for 16 h (f). Scale bars, 20 µm. Data in (f) were quantified (g). At least 100 nuclei were quantified for per condition (e). h Western blot showed that the pRPA levels were decreased in SIRT5-silenced CRC cells after supply of exogenous nucleosides. HU was used as a positive control. i–l Exogenous supplementation with the four nucleosides restored DNA synthesis in CRC cells after SIRT5 knockdown. EdU assay involving immunofluorescent staining (i) and flow cytometry (k). Scale bars, 20 µm. The data in (i and k) were quantified and analyzed in (j) and (l) respectively. (n = 3 biologically independent experiments). Data are presented as mean values ± SEM. Statistical significance was calculated using one-way ANOVA corrected with Tukey’s multiple comparisons test. ns not significant. Source data are provided as a Source Data file.