Basbous J , Aze A , Chaloin L , Lebdy R , Hodroj D , Ribeyre C , Larroque M , Shepard C , Kim B , Pruvost A , Moreaux J , Maiorano D , Mechali M , Constantinou A .

R01 AI136581 NIAID NIH HHS , R01 AI150451 NIAID NIH HHS

             DNA repair

	Figure 1. Depletion of DHP impairs the proliferation of epithelial cancer cells. (A) Schematic representation of the pyrimidine degradation pathway. (B) DHP was probed by western blotting in the indicated transformed cells. When indicated, DHP was knocked down using anti-DHP siRNA or shRNA molecules with distinct target sequences. Ponceau staining was used as loading control. * non-specific signal. One representative experiment is shown from three to six biological replicates. (C) U-2 OS cells were transfected with control or anti-DHP siRNA (siDHP-sp) and their viability was assessed during 4 days using the MTT cell growth assay. Mean viability is representative of three independent biological replicates. Error bars represent ±S.D. (D) Colony-forming assay of U-2 OS cells after transfection with control or anti-DHP siRNA (siDHP-1). A representative image is shown. An histogram represents the quantification of colony formation. Data shown are averages over three independent biological replicates with two technical replicates for each. Error bars represent ±S.D. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. Bottom panel: the efficiency of DHP knockdown was assessed by western blotting. (E) Histogram representing the percentage of U-2 OS cells, 72 h after transfection with control or anti-DHP siRNA (siDHP-1) in G0/G1, S and G2/M phases. Data shown are averages over three independent biological replicates. Error bars represent ±S.D. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001.
	Figure 2. Suppression of DHP interferes with replication fork progression and induces activation of DNA damage responses. (A) Experimental scheme: 72 h after transfection with control or anti-DHP siRNA (siDHP-1), cells were labeled with two consecutive pulses of 30 min with CldU and IdU, as indicated. DNA was stretched out on glass slides and newly synthesized DNA was revealed by immunofluorescence. Graphic representation of replication track lengths in U-2 OS cells co-transfected with control or anti-DHP siRNA (siDHP-1) along with control plasmid or a plasmid encoding siRNA-resistant DHP. The bar dissecting the data points represents the median of 100 tracts length from one biological replicate. Differences between distributions were assessed with the Mann–Whitney rank sum test. P-values: *** < 0.0001. (B) Whole-cell extracts from control and DHP knockdown U-2 OS cells (siDHP-1) were analyzed by western blotting with the indicated antibodies. One representative experiment is shown from more than three biological replicates (C) RPA32 immunofluorescence staining of control and DHP knockdown U-2 OS cells (siDHP-1). DNA was stained by Hoechst. Bars indicate 10 μm. Bottom panel: Quantification of the percentage of RPA32 foci positive cells in a population of 100 cells. Data from three independent biological replicates are represented as mean ± S.D. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. (D) Immunofluorescence staining of Ser33 phospho RPA32 in control and DHP knockdown U-2 OS cells (siDHP-1). DNA was stained by Hoechst. Bars indicate 10 μm. Bottom panel: Histogram representing the percentage of Ser33 pRPA32 foci positive cells in a population of 100 cells. Data from three independent biological replicates are represented as mean ± S.D. (100 cells were counted per experiment). P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. (E) Control and DHP knockdown U-2 OS cells (siDHP-1) were uniformly labeled with BrdU before immunofluorescence staining in native conditions with an anti-BrdU antibody. DNA was stained by Hoechst. Bars indicate 10 μm. Right panel: Histogram representation of the percentage of ssDNA positive cells. Values are the mean ± S.D of three independent biological replicates (100 cells were counted per experiment). P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. (F) Western blotting analysis with the indicated antibodies of whole-cell extracts from control and DHP knockdown U-2 OS cells (siDHP-1) complemented or not with a siRNA-resistant DHP cDNA, as indicated. One representative experiment is shown from three biological replicates.
	Figure 3. Accumulation of dihydropyrimidines induces DNA replication stress. (A) The concentrations of dihydrouracil and uracil were measured in U-2 OS cells transfected with control or anti-DHP siRNA (siDHP-1). The ratio of molar concentrations between the two metabolites in each sample is presented. Data from three independent biological replicates, with three technical replicates for each, are represented as mean ± S.E.M. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. (B) Replication tracks were labeled with two consecutive pulses of 30 min with CldU and IdU in U-2 OS cells transfected with the indicated siRNAs. Graphic representations of replication track lengths measured in μm (y-axis). The bar dissecting the data points represents the median of 100 tracts length from one biological replicate. Differences between distributions were assessed with the Mann–Whitney rank sum test. P-values: *** < 0.0001. (C) Western blot analysis with the indicated antibodies of whole cell extracts from U-2 OS transfected with anti-DHP and anti-DPD siRNAs, as indicated, Ponceau staining was used as control of protein loading and transfer. * non-specific band. One representative experiment is shown from two biological replicates. (D) RPA32 immunofluorescence staining of U-2 OS cells transfected with the indicated siRNAs. Bars indicate 10 μm. DNA was stained by Hoechst. Bottom panel: Histogram representation of the percentage of RPA32 foci-positive cells in a population of 100 cells. Data from three independent biological replicates are represented as mean ± S.D. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001.
	Figure 4. Dihydropyrimidines induce transcriptional stress and yield abnormal DNA replication intermediates. (A) Graphic representation of global transcriptional activity visualized by 5-ethynyl uridine (EU) incorporation. U-2 OS cells transfected with control and anti-DHP siRNA (siDHP-1) were labeled with EU for 20 min before fixation. The EU intensity of 100 cells from two independent biological replicates was measured by fluorescence microscopy. The bar dissecting the data points represents the median of EU intensity. Differences between distributions were assessed with the Mann–Whitney rank sum test. P-values: *** < 0.0001. (B) Immunofluorescence staining with S9.6 and nucleolin antibodies of DHP-depleted or control U-2 OS cells (siDHP-sp). DNA was stained by Hoechst. Bars indicate 10 μm. Right panel: The graph shows the median of S9.6 signal intensity par nucleus after nucleolar signal removal. More than 1000 cells from two independent biological replicates were considered. Differences between distributions were assessed with the Mann–Whitney rank sum test. P-values: *** < 0.0001. (C) Left panel: Experimental scheme. DNA synthesis reactions (control: DMSO; DHU: 15 mM) were pulse-labeled for 30 min with α-[32P]-dCTP at the indicated times during the course of a 2-h reaction. Replication products were purified and resolved by electrophoresis through a 1.2% agarose gel in denaturing conditions. (*) abnormal replication intermediate. One representative experiment is shown from two biological replicates. (D) Experimental scheme: Sperm nuclei were added to Xenopus egg-extract (in presence or not of 7.5 mM DHU dissolved in water) and incubated at 23°C to allow origins firing and replication initiation. After 30 min incubation, the firing of new replication origins was blocked with roscovitine (0.5 mM). Replicating nuclei were then isolated after 60 min of incubation and transferred to a second extract (restarting extract) supplemented with roscovitine (0.5 mM) and Geminin (60 mM) to block the firing and the assembly of novel origins, respectively. DNA synthesis reactions were pulse-labeled with α-[32P]-dCTP during incubation in the second extract. (E) Replication products were resolved by 1% alkaline agarose gel electrophoresis and revealed by autoradiography. Lanes 1–4: Mock treated extracts; Lanes 5–8: incubation in the first extract was performed in the presence of 7.5 mM DHU. Lanes 10–12 serve as positive controls: after 30 min incubation in the first extract, DNA synthesis was blocked with aphidicolin (100 ng/μl). Right panel: Histogram representing the quantification of the gel by image J of replication products (arbitrary unit). One representative experiment is shown from two biological replicates (See Supplementary Figure S4A).
	Figure 5. Dihydromyricetin induces DNA replication stress. (A) IC50 determination of dihydromyricetin for DHP (0.2 μM) using dihydrouracil (50 μM) as a substrate. One representative experiment is shown from three biological replicates. (B) Molar ratios of dihydrouracil versus uracil measured in U-2 OS cells treated with 20 μM dihydromyricetin for 16 h. Data from three independent biological replicates, with three technical replicates for each, are represented as mean ± S.E.M. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. (C) Western blot analysis with the indicated antibodies of U-2 OS whole-cell extracts treated with dihydromyricetin for 48 h at the indicated concentrations. One representative experiment is shown from three biological replicates. (D) RPA32 immunofluorescence staining of U-2 OS cells treated with DMSO or 20 μM dihydromyricetin for 16 h. Bars indicate 10 μm. DNA was stained by Hoechst. Bottom panel: Histogram representation of the percentage of RPA32 foci-positive cells in a population of 100 cells. Data from three independent biological replicates are represented as mean ± S.D. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. (E) Graphic representation of replication track lengths measured in μm (y-axis) in control and U-2 OS cells treated with 20 μM of dihydromyricetin for 16 h. The bar dissecting the data points represents the median of 100 tracts length from one biological replicate. Differences between distributions were assessed with the Mann-Whitney rank sum test. P-values: *** < 0.0001. (F) U-2 OS cells were transfected with control or anti-DPD siRNA and exposed to increasing concentrations of dihydromyricetin for 2 days. Cell viability was estimated using Cell Titer-Glo assay. Mean viability is representative of experiments performed in triplicate. Error bars represent ± S.E.M. (G) Western blot analysis with the indicated antibodies of whole cell extracts from U-2 OS transfected with anti-DPD siRNA and treated or not with 20 μM of dihydromyricetin for 24 h. One representative experiment is shown from two biological replicates.
	Figure 6. Dihydropyrimidines metabolites induce DPCs. (A) The chromatin fraction of control and DHP knockdown U-2 OS cells (siDHP-1) was subjected to western blot analysis with the indicated antibodies. Histone H3 was used as loading control. One representative experiment is shown from two biological replicates. (B) Chromatin extracts from nuclei incubated in control and DHU (7.5 mM) containing extracts for 60 and 120 min were subjected to western blot analysis with the indicated antibodies. Histone H3 was used as loading control. One representative experiment is shown from two biological replicates. (C) Total DPC levels in U-2 OS cells transfected with control or anti-DHP siRNA (siDHP-1) visualized by silver staining. Right panel: Histogram representing the quantification of DPC levels normalized to total DNA amount by image J. Three independent biological replicates are averaged in the bar graphs. Error bars represent ± S.D. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. (D) Total DPC levels in U-2 OS cells treated or not with 1 mM FA for 2 h visualized by silver staining. Right panel: Histogram representing the quantification of DPC levels normalized to total DNA amount by image J. Three independent biological replicates are averaged in the bar graphs. Error bars represent ± S.D. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. (E) Total DPC levels after U-2 OS cells treatment with DMSO or 5 μM of Dihydromyricetin for 16 h visualized by silver staining. Right panel: Histogram representing the quantification of DPC levels normalized to total DNA amount by image J. Three independent biological replicates are averaged in the bar graphs. Error bars represent ± S.D. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. (F) Western blot analysis of crosslinked DNA polymerase η in total DPC extracts from U-2 OS cells transfected with control or anti-DHP siRNA (siDHP-1) and the corresponding DNA quantification. One representative experiment is shown from two biological replicates. (G) Slot-blot showing crosslinked DNA polymerase η in total DPC extracts from U-2 OS cells treated with 20 μM dihydromyricetin for 16 h and the corresponding DNA quantification. (H) Pie chart representation of gene ontology analyses using the panther Classification system (http://www.pantherdb.org/on). Theoretical relates to gene ontology classification expected from random sampling of the proteome. Observed is the classification of proteins enriched at least 10-folds in DHP-depleted cells versus control cells. Proteins selected for the analysis were identified in three independent experiments.
	Figure 7. FANCM promotes cellular tolerance to dihydromyricetin. (A) U-2 OS cells were transfected with the indicated siRNAs and exposed to increasing concentrations of dihydromyricetin for 2 days. Cell viability was estimated using Cell Titer-Glo assay. Mean viability is representative of experiments preformed in triplicate. Error bars represent ±S.D. Bottom panel: the efficiency of FANCM, FANCD2 or FANCA knockdown was assessed by western blotting. (B) U-2 OS cells were transfected with FANCM siRNA and cell viability was assessed as described in A. Mean viability representative of quadruplicates. Error bars represent ±S.D. (C) Model: The accumulation of Dihydropyrimidines in cancer cells induces DNA replication and transcriptional stress via the formation of DPCs.

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