Fig 2: Comparison of NUDIX protein activity with nucleotide substrates.(a) Nucleotide substrate (50 µM) was incubated with 5–500 nM NUDIX protein depending on enzyme. Hydrolysis was monitored by detecting phosphate generated. The depicted data are representative of two independent experiments showing the same result. Data are presented as v (hydrolysed substrate (µM) per minute) per [enzyme] (µM). (b) Saturation curves and kinetic parameters of MTH1- and NUDT15-mediated hydrolysis of 8-oxo-dGTP (left) and dGTP (right). NUDT15 (8 nM) or MTH1 (0.25 nM) was incubated with 8-oxo-dGTP at concentrations ranging from 0 to 100 µM in assay buffer, and initial rates were determined in duplicate. Inset highlights NUDT15 activity on a smaller activity scale. NUDT15 (8 nM) and MTH1 (2 nM) were incubated with dGTP in assay buffer ranging from 0 to 400 µM, and initial rates were determined in duplicate. Data are presented as v (hydrolysed substrate (µM) per second) per [enzyme] (µM), and are representative of data collected from at least two independent experiments. (c) Kinetic parameters of MTH1 and NUDT15 for dGTP and 8-oxo-dGTP hydrolysis. The Michaelis–Menten equation was applied to saturation curves using the GraphPad Prism software and kinetic parameters were calculated. Data presented are average±s.d. from two independent experiments. (d) HPLC chromatograms showing the activity of NUDT15 and MTH1 against 8-oxo-dGTP and dGTP. The depicted data are representative of three independent experiments and show that MTH1 rapidly hydrolyses 8-oxo-dGTP; however, no significant activity is observed with NUDT15. Both MTH1 and NUDT15 can hydrolyse dGTP.

Fig 3: Co-depletion of OGG1 in A549 lung adenocarcinoma cells protects against MTH1 depletion-induced DNA breaks and p53-induced senescence.A. Simultaneous lentiviral co-transduction and selection schemes employed in this experiment. The pLKO.shMTH1 construct is under hygromycin (h) selection and the pLKO.shOGG1 construct is under puromycin (p) selection. Control samples consist of co-transduced puromycin- and hygromycin-selectable pLKO.shGFP constructs. B. Immunoblot showing protein levels of OGG1 and MTH1 under the various individual or co-selection schemes denoted in (A) in the shRNA co-transduced cells. Actin is shown as the loading control. C. Quantitation of actin-normalized OGG1 and MTH1 protein expression levels from (B) under the different selection schemes. Normalized quantifications are based on pixel densities of immunoblot bands obtained via ImageJ. D. Alkaline comet assay. Representative olive tail moments (10× magnification) are indicated for the types of comets that were scored (no/medium/long tails) in the indicated cultures. Error bars (±SD) and p-values from an unpaired Student t-test are shown from two independent experiments. E. Senescence-associated beta-galactosidase (SA-beta-gal) activity assay. Cell senescence was assessed in the indicated cultures. Positive staining was quantified in approximately 50 cells per condition. Data represent two experimental replicates. Error bars (±SD) and p-values from an unpaired Student's t-test significance are shown. F. Immunoblot showing p53 and actin (loading control) protein levels in the indicated samples.

Fig 4: Lung adenocarcinomas show a selective disadvantage against high OGG1 levels in the context of low MTH1 (NUDT1) status.A. MTH1 and OGG1 mRNA expression levels in paired normal/tumor non-small cell lung cancer (NSCLC) patient-derived tissues. Each number represents a specimen pair from a distinct patient. Normalized tumor values (each sample value divided by the value from its normal counterpart, set to 1 as per the horizontal line) are shown for OGG1 and MTH1. B. Frequency (percentage) of OGG1 vs. MTH1 mutations or copy number alterations (CNA). Gene amplifications (red), mutations (green), and deletions (blue) are shown for lung adenocarcinoma specimens from 816 patients. Graphs are derived from The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LUAD) datasets at cbioportal.org. C. Correlations between MTH1 and OGG1 levels in the above LUAD dataset. Spearman and Pearson correlation coefficients are shown (generated automatically at cbioportal.org). Expression levels are indicated by z-scores, which represent the numbers of standard deviations above or below the mean raw gene expression. The black gridlines demarcate low MTH1 (Lo M, z-scores 0 or lower) vs. robust OGG1 (Hi O, z-scores 2 or higher) levels and the red gridlines demarcate low OGG1 (Lo O, z-scores 0 or lower) vs. robust MTH1 (Hi M, z-scores 2 or higher) levels. Normal (Nrml) values are designated for z-scores greater than 0 and less than 2. Quadrants made by the intersecting gridlines are labeled according to the legend on the right of the graph. Note the significant representation of tumors with low OGG1 and low MTH1 (Lo O/Lo M) and the lack of Hi O/Lo M tumors. D. Immunoblot showing OGG1 and MTH1 levels in H23 xenograft tumors. Flash-frozen tumors were lysed and total protein lysate probed against the indicated antibodies, with actin as the loading control. Tumor sizes (in mm3) corresponding to the samples are indicated below the immunoblot lanes. Quantitated OGG1 levels (normalized to their actin signals) from the shGFP and shMTH1 tumors are shown below the immunoblot (error bars indicated ±SEM). Note that the variation in tumor OGG1 levels observed in the shGFP samples are collapsed to the low OGG1/low MTH1 (Lo O/Lo M) category in the shMTH1 samples. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: Determination of rearrangement signatures in gene knockouts. a The rearrangement spectra of parental clones and DNA repair gene knockouts are represented by a 10-channel profile that takes the type and length of rearrangements into account. The rearrangement spectrum of each sample is shown in Supplementary Fig. 4. Error bars were referred to as standard error of means (n = 7). b Distribution of bootstrapped rearrangement spectra of parental clone (pink) and subclones (green) of the knockouts. EXO1, FANCC and NUDT1 knockouts show significant changes in their rearrangement profiles. c Identification of elevated rearrangement numbers in knockouts. EXO1 and FANCC knockouts show high number of rearrangements (p value <= 0.01), while NUDT1 has a p value of 0.0105, which is at the border of our threshold. To be conservative, NUDT1 is not determined to have a rearrangement signature. d Rearrangement signature of EXO1 and FANCC. The rearrangement signature associated with knockout of EXO1 is characterised by median tandem duplications (10 kb–1 Mb). The rearrangement signature associated with knockout of FANCC is characterised by short deletions (1–10 kb), deletions and tandem duplications of 1–10 kb and 10 kb–1 Mb