Journal: Nucleic Acids Research

Article Title: DNA-PK cs promotes chromatin decondensation to facilitate initiation of the DNA damage response

doi: 10.1093/nar/gkz694

Figure Lengend Snippet: Generation and initial characterization of the DNA-PK cs kinase-dead (KD) HCT116 cell line. ( A ) Basic schematic of the method used to generate the HCT116 DNA-PK cs kinase dead (KD/−) cell line. ( B ) Expression level of DNA-PK cs in HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cells as assessed by western blotting. ( C ) Measurement of DNA-PK cs in vitro kinase activity. Nuclear extracts from the HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cells were examined for their ability to phosphorylate a biotin-tagged H2AX peptide. H2AX phosphorylation was observed in the −/− cell line and this was subtracted from the other samples’ readouts. The 100% kinase activity was normalized using the +/+ cell lysate results. The data are presented as the mean ± SD from three individual experiments. ( D ) Measurement of DNA-PK cs in vivo kinase activity. The HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cell lines were mock-treated or γ-irradiated with a dose of 10 Gy and allowed to recover for 30 min. Cell extracts were prepared and western blot analysis was performed to assess autophosphorylation of DNA-PK cs at serine 2056. Immunoblotting of Ku70 and Ku80 were used as loading controls. ( E ) The interaction between DNA-PK cs and the Ku70/80 heterodimer is not affected by inactivating the kinase activity of DNA-PK cs . DNA-PK cs was immunoprecipitated from the HCT116 +/+, +/−, −/−, and KD/− cell lines 5 min after being irradiated with 10 Gy of γ-rays. The immunoprecipitates were analyzed by western blotting with anti-DNA-PK cs , Ku80, and Ku70 antibodies. Tubulin was used as a loading control for the input of each immunoprecipitation assay.

Article Snippet: Briefly, the HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with 10 Gy and allowed to recover for 10 min. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, In Vitro, Activity Assay, Phospho-proteomics, In Vivo, Irradiation, Immunoprecipitation, Control

Journal: Nucleic Acids Research

Article Title: DNA-PK cs promotes chromatin decondensation to facilitate initiation of the DNA damage response

doi: 10.1093/nar/gkz694

Figure Lengend Snippet: DNA-PK cs kinase activity is important for cell survival and DSB repair following DSB induction. ( A ) Colony formation assays were performed to compare radiation sensitivities of the HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cell lines. Cells lines were left cycling and irradiated at the indicated doses and plated for analysis of survival and colony-forming ability. The data is presented as mean ± SD from three independent experiments. ( B ) DSB repair proficiency of HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− was evaluated using neutral Comet assay. Cells were irradiated with 10 Gy of IR, allowed to recover for 30 min, harvested, and then Comet assays were performed. Tail moment values for >400 mock treated and irradiated cells were calculated and plotted via a distribution dot plot. ( C ) Dynamic 53BP foci distribution in HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cells after IR. The cells were irradiated with 1 Gy of γ-rays and 53BP1 foci formation and resolution was assessed 0.5, 1, 2, 4 and 8 h later. Data were normalized to the foci number enumerated at 30 min post IR. Remaining foci number per time point were calculated and plotted. Error bars denote SEM of three independent experiments. ( D ) NHEJ-mediated and ( E ) HR-mediated DSB repair were evaluated in HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cells using GFP-based reporter assays. The data were presented as Mean ± SEM with P -values from at least three biological repeats.

Article Snippet: Briefly, the HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with 10 Gy and allowed to recover for 10 min. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions.

Techniques: Activity Assay, Irradiation, Neutral Comet Assay

Journal: Nucleic Acids Research

Article Title: DNA-PK cs promotes chromatin decondensation to facilitate initiation of the DNA damage response

doi: 10.1093/nar/gkz694

Figure Lengend Snippet: DNA-PK cs initiates phosphorylation of H2AX and KAP1 after IR. ( A ) IR-induced phosphorylation of KAP1 and H2AX is attenuated in the KD/− cell line at early time points. HCT116 DNA-PK cs +/−, −/−, and KD/− cell lines were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 1, 3, or 5 min. Whole cell lysates were obtained and immunoblotting was performed to assess the phosphorylation status of DNA-PK cs at serine 2056, ATM at serine 1981, KAP1 at serine 824, and H2AX at serine 139. Tubulin was used as a loading control. ( B ) H2AX and KAP1 are phosphorylated in ATM-deficient cells (AT5) after IR, which is dependent on DNA-PK cs . AT5 cells were mock-treated or incubated for 2 h prior to irradiation with 10 μM NU7441 to inhibit DNA-PK cs and then AT5 cells and ATM-expressing AT5 cells (AT5+ATM) were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 3, 10, or 30 min. Whole cell lysates were obtained and immunoblotting was performed to assess the phosphorylation status of DNA-PK cs at serine 2056, KAP1 at serine 824, and H2AX at serine 139. Tubulin was used as a loading control. ( C – E ) Time courses of IR-induced KAP1 and H2AX phosphorylation in the HCT116 DNA-PK cs +/−, −/−, and KD/− cell lines. The cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 1, 3, 5, 10, 15, 30, or 60 min. (C) Whole cell lysates were obtained and immunoblotting was performed to assess the phosphorylation status of DNA-PK cs at serine 2056, KAP1 at serine 824, and H2AX at serine 139. Tubulin was used as a loading control. All immunoblots for each protein shown in (C) come from the same exposure of a single film. The relative phosphorylation level of H2AX and KAP1 at each time point are shown in (D) and (E), respectively. Following quantification of the protein levels, the relative level of H2AX and KAP1 phosphorylation were calculated by taking γH2AX/H2AX and KAP1 S824/KAP1 and then normalizing each time point to value of γH2AX/H2AX and KAP1 S824/KAP1 at 1 h after IR in the (+/−) control cell line. The data are presented as the mean ± SEM from three independent experiments. The P -values were generated by comparing +/− with −/− or KD/− with Student t -test. *, P < 0.05; **, P < 0.01. All data (A–C) are a representative image of three independent experiments.

Article Snippet: Briefly, the HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with 10 Gy and allowed to recover for 10 min. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions.

Techniques: Phospho-proteomics, Irradiation, Western Blot, Control, Incubation, Expressing, Generated

Journal: Nucleic Acids Research

Article Title: DNA-PK cs promotes chromatin decondensation to facilitate initiation of the DNA damage response

doi: 10.1093/nar/gkz694

Figure Lengend Snippet: Chromatin decondensation in response to DNA damage at early time points requires DNA-PK cs kinase activity. ( A ) IR-induced chromatin relaxation is attenuated in the HCT116 DNA-PK cs KD/− and −/− cells. The HCT116 DNA-PK cs +/−, −/−, and KD/− cell lines were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Chromatin decondensation was then determined by examining micronuclease (MNase) accessibility. Nuclei were processed and the DNA was visualized by resolving it via agarose gel electrophoresis. Panels on the right show quantified signal as the percent of total for each lane across a distance from the well to the end of gel. ( B ) ATM kinase activity is not required for the initial chromatin relaxation after IR-induced DNA damage. The HCT116 DNA-PK cs +/− and KD/− cell lines were mock-treated or incubated for 2 h prior to irradiation with 10 μM KU60019 to inhibit ATM and then the cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Samples were processed and quantified as described in (A). ( C ) Inhibition of DNA-PK cs suppresses IR-induced chromatin decondensation in ATM-deficient AT5 cells. The AT5 cells were mock-treated or incubated for 2 h prior to irradiation with 10 μM NU7441 to inhibit DNA-PK cs and then the cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Samples were processed and quantified as described in (A). All data (A–C) are a representative image of three independent experiments.

Article Snippet: Briefly, the HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with 10 Gy and allowed to recover for 10 min. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions.

Techniques: Activity Assay, Irradiation, Agarose Gel Electrophoresis, Incubation, Inhibition

Journal: Nucleic Acids Research

Article Title: DNA-PK cs promotes chromatin decondensation to facilitate initiation of the DNA damage response

doi: 10.1093/nar/gkz694

Figure Lengend Snippet: DNA-PK cs catalytic activity facilitates the initial recruitment of the DDR machinery to DSBs. ( A ) Recruitment of HR and NHEJ factors to the chromatin after IR is attenuated in the HCT116 DNA-PK cs KD/− cell line. HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Subsequently, soluble nuclear and the chromatin-enriched fractions were isolated for immunoblotting to assess the recruitment of the proteins listed in the figure to the chromatin after irradiation. ( B ) IR-induced focus formation of MDC1 is attenuated in the HCT116 DNA-PK cs KD/− cell line. The HCT116 DNA-PK cs +/−, −/−, and KD/− cell lines were mock-treated or irradiated with a dose of 1 Gy and MDC1 foci formation was assessed 5 and 10 min later. MDC1 focus formation was examined in at least 50 cells and the number of MDC1 IR-induced foci per nucleus is shown. ****, P value < 0.0001. ( C – F ) Recruitment of GFP-tagged (C) NBS1, (D) EXO1, (E) XLF, and (F) XRCC4 to laser-generated DSBs is attenuated in the HCT116 DNA-PK cs KD/− cell line compared to the +/− cells. Relative fluorescent intensity of GFP-tagged NBS1, EXO1, XLF, and XRCC4 are presented as the mean ± SEM. *, P value < 0.05.

Article Snippet: Briefly, the HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with 10 Gy and allowed to recover for 10 min. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions.

Techniques: Activity Assay, Irradiation, Isolation, Western Blot, Generated

Journal: Cell reports

Article Title: mTORC1 regulates the pyrimidine salvage pathway by controlling UCK2 turnover via the CTLH-WDR26 E3 ligase

doi: 10.1016/j.celrep.2024.115179

Figure Lengend Snippet: (A) Analysis of the previously reported AHA degradomics data is presented as a volcano plot of the log 10 -transformed p value versus the log 2 -transformed ratio of Torin1 treated/untreated (UT) conditions for WT HEK293T cells. UCK2 degradation is accelerated after mTORC1 inhibition for 12 h using Torin1 (250 nM). p values were calculated by two-sided Welch’s t test (adjusted to 1% false discovery rate for multiple comparisons). A total of 8,644 proteins were quantified. n =3 biological replicates. (B) Line histograms of UCK2 and UCK1 decay (data from WT, ATG7 −/− , and FIP200 −/− HEK293T cells all combined) with or without Torin1 treatment were extracted from the previously reported dataset. n = 6 for each data point, mean ± SD. (C) Immunoblot of HEK293T extracts treated with the indicated inhibitors: Torin1 (250 nM), MLN7243 (100 nM), and BTZ (500 nM). (D) HEK293T cell lysates treated with Torin1 or RM6 for the indicated time were subjected to immunoblot analysis using the specified antibodies. Torin1 (250 nM) inhibits both mTORC1 and mTORC2, whereas RM6 (3 nM) selectively inhibits mTORC1. (E) Amino acid (AA) deprivation leads to reduced UCK2 levels. (F) Quantification for (D) and (E). n ≥ 4 biological replicates. Data are represented by mean ± SD. **** p < 0.0001. (G) MFE296 and Jurkat cells show a similar reduction in UCK2 upon mTORC1 inhibition by RM6 (3 nM). (H) The decrease of UCK2 during mTORC1 inhibition is post-translationally regulated, as indicated by the AHA degradomics. Cells were treated with 1-μM CHX and 3-nM RM6 for 16 h. CHX: 1 μM, RM6: 3 nM. (I) Torin1 treatment (200 nM) does not affect relative UCK2 mRNA levels. (J) UCK2-mEGFP knockin cell line generation using CRISPR-Cas9. Genotyping data is shown for K/I validation. (K) Histogram of GFP intensity when UCK2-mEGFP knockin cells were treated with RM6 (1 nM) in the presence or absence of MLN7243 (100 nM) for 16 h before flow cytometry analysis. n = 2,347 cells. (L) Bar graph representing mean ± SD of the biological triplicate experiments performed as in (K) using HEK293T and HCT116 cells. *** p < 0.001. (M) Flow cytometry analysis of UCK2-mEGFP cells shows that 1-μM CHX and joint 1-μM CHX/3-nM RM6 treatment reduce UCK2 levels by 25% and 50%, relative to the untreated control. Mean ± SD from n = 3 biological replicates. ** p < 0.01. (N) Proposed model of UCK2 degradation under mTOR on and off conditions.

Article Snippet: Human: HCT116 , ATCC , CCL-221; RRID:CVCL_0291.

Techniques: Transformation Assay, Inhibition, Western Blot, Knock-In, CRISPR, Biomarker Discovery, Flow Cytometry, Control

Journal: Cell reports

Article Title: mTORC1 regulates the pyrimidine salvage pathway by controlling UCK2 turnover via the CTLH-WDR26 E3 ligase

doi: 10.1016/j.celrep.2024.115179

Figure Lengend Snippet: (A) Imaging analysis of 293T and HCT116 UCK2-mEGFP homozygous K/I cells displays heterogeneous UCK2 subcellular localization across the clonal cells. 1 exemplifies N < C, 2 exemplifies N = C, and 3 exemplifies N > C. Scale bar, 10 μm. (B) Quantification of (A). N < C: cytoplasmic accumulation, N = C: equal distribution between the nucleus and cytoplasm, N > C: nuclear accumulation. n = 3 image sections, mean ± SD shown. Eighty-six cells for HEK293T and 148 cells for HCT116 were quantified. (C) Live cell imaging analysis of HCT116 UCK2-mEGFP K/I cells suggests that UCK2 localization changes as the cell cycle progresses. (D) Double thymidine blocking scheme to synchronize cells at the early S phase. (E) Cells synchronized with double thymidine block enter mitosis 8–10 h following release. (F) A representative image of the UCK2-mEGFP cells following the thymidine release indicates that UCK2 is concentrated in the nucleus at the early S phase. Scale bar, 10 μm. (G) HEK293T UCK2-mEGFP cells underwent double thymidine blocking as in (D), and the nuclear-cytosolic distribution of UCK2 was analyzed in individual cells from their post-mitotic point (0 h) and every hour for 12 h thereafter. n = 42 cells were quantified. N < C: cytoplasmic, N = C: equal distribution, N > C: nuclear. (H) The 16-h RM6 treatment changes the distribution of UCK2 in MAEA-dependent manner using HEK293T UCK2-mEGFP K/I cells. The population of cells showing N > C UCK2 distribution increases in WT, but not MAEA −/− , cells after RM6 treatment. Scale bar, 20 μm. (I) The images in (H) are represented using a LUT (look-up table) to show the pixel brightness. Identical exposure time and laser intensity were applied for microscopic analysis. Scale bar, 20 μm, pixel intensity: A.U. (J) The quantification of n > 180 cells in (H) is shown. Mean ± SD of n > 13 image sections.

Article Snippet: Human: HCT116 , ATCC , CCL-221; RRID:CVCL_0291.

Techniques: Imaging, Live Cell Imaging, Blocking Assay

Journal: Cell reports

Article Title: mTORC1 regulates the pyrimidine salvage pathway by controlling UCK2 turnover via the CTLH-WDR26 E3 ligase

doi: 10.1016/j.celrep.2024.115179

Figure Lengend Snippet: (A) Microscopy analysis of the UCK2-mEGFP = K/I cells with WT UCK1, UCK1 −/− , or UCK1 −/− over-expressing mCherry-UCK1. Depletion of UCK1 results in cytoplasmic accumulation of UCK2, while over-expression of mCherry-UCK1 leads to nuclear accumulation of UCK2. mCh, mCherry. Scale bar, 20 μm. (B) Quantification of (A). n > 250 cells. Mean ± SD of n > 4 image sections is shown. (C) Flow cytometry analysis of the HEK293T UCK2-mEGFP K/I cells (WT, UCK1 −/− , or UCK1 V5 over-expression in WT background) shows that over-expressing UCK1 hinders efficient turnover of UCK2. n = 3 biological replicates. O/E, over-expression. * p < 0.05, **** p < 0.0001. (D) UCK2-mEGFP K/I cells with WT UCK1, UCK1 −/− , or UCK1 −/− over-expressing mCherry-UCK1 were treated or left untreated with RM6 for 16 h before live-cell imaging analysis. Scale bar, 20 μm. See also . (E) Generation of HEK293T and HCT116 cells expressing endogenous UCK2-HA via CRISPR-Cas9 approach. (F) Immunoprecipitation of HEK293T UCK2-HA K/I cells detects strong interactions between endogenous UCK1 and UCK2 proteins. asterisk: UCK2-HA detected by the polyclonal UCK1 antibody. (G) HEK293T UCK2-HA K/I cells were subjected to formaldehyde crosslinking (0.5%, 1 h) before immunoblotting. Anti-HA antibody-reactive ladders suggest that UCK2 forms oligomers in cells. F.A., formaldehyde. (H) Immunoprecipitation of HEK293T UCK2-HA cell lysates following formaldehyde crosslinking (0.5%, 30 min). The elute of the crosslinked sample shows strong enrichment of monomeric and potentially dimeric UCK1 complex, suggesting their binding to UCK2 monomer and dimers. *UCK2-HA detected by the polyclonal UCK1 antibody. (I) Proposed model of UCK2 and UCK1 hetero oligomerization and their role in UCK2 subcellular localization.

Article Snippet: Human: HCT116 , ATCC , CCL-221; RRID:CVCL_0291.

Techniques: Microscopy, Expressing, Over Expression, Flow Cytometry, Live Cell Imaging, CRISPR, Immunoprecipitation, Western Blot, Binding Assay

Journal: Cell reports

Article Title: mTORC1 regulates the pyrimidine salvage pathway by controlling UCK2 turnover via the CTLH-WDR26 E3 ligase

doi: 10.1016/j.celrep.2024.115179

Figure Lengend Snippet: (A) Schematic of heavy isotope tracing. L-glutamine- N serves as a label for de novo pathway products, whereas uridine- 13 C 5 labels metabolites synthesized through the salvage pathway. (B) Immunoblot comparing UCK2 protein levels in the cell lines used in isotope tracing. (C) Quantification of UCK2 levels between WT and MAEA −/− cells as in (B). MAEA −/− cells express 30% higher UCK2 levels than WT. ** p < 0.01. (D and E) Relative abundance of UDP and UTP containing 15 N ( de novo product), 13 C 5 (salvage product), and no heavy isotope (unlabeled). Mean ± SD of n =4 biological replicates. *** p < 0.001, **** p < 0.0001. (F) Cells with higher UCK2 levels may demonstrate more efficient metabolism of pyrimidine analog prodrugs into their cytotoxic forms. (G) Cell survival of WT, MAEA −/− , and UCK2 over-expressing HEK293T cells in response to 72 h of 5-azacytidine treatment. MAEA −/− cells were twice as sensitive as WT cells, whereas UCK2 over-expressing cells had a 160-fold decrease in their IC 50 . Mean ± SD of n = 6 biological replicates. * p < 0.05, ** p < 0.01, **** p < 0.0001. (H) Cell survival at 72 h after treatment with 5-azacytidine (5-AzaCd) or 5-fluorouridine (5-FUd) with or without dTAGv1 (100 nM) in HCT116 UCK2-FKBP12 F36V K/I cells. dTAGv1-mediated degradation of UCK2 results in 156-fold resistance to 5-FUd and 1.6-fold to 5-AzaCd. Mean ± SD, n = 12 biological replicates.

Article Snippet: Human: HCT116 , ATCC , CCL-221; RRID:CVCL_0291.

Techniques: Synthesized, Western Blot, Expressing

Journal: Cell reports

Article Title: mTORC1 regulates the pyrimidine salvage pathway by controlling UCK2 turnover via the CTLH-WDR26 E3 ligase

doi: 10.1016/j.celrep.2024.115179

Figure Lengend Snippet:

Article Snippet: Human: HCT116 , ATCC , CCL-221; RRID:CVCL_0291.

Techniques: Recombinant, Staining, Protease Inhibitor, Plasmid Preparation, Gel Extraction, DNA Purification, Sequencing, Software